BACTERIOPHAGE PHENOMENA 


4 


BY 

ANN GAYLER KUTTNER 


From the Department of Bacteriology, College of Physicians and Burgeons 



Reprinted from 

Journal of Bacteriology, Vol. VIII, No. 1, 
January, 1923 











'v * y i 








g, (Si .Wt«A - 2 - 



Reprinted from Journal op Bacteriology 
Vol. VIII, No. 1, January, 1923 


BACTERIOPHAGE PHENOMENA 1 

ANN GAYLER KUTTNER 

From the Department of Bacteriology, College of Physicians and Surgeons 
Received for publication June 6, 1922 

INTRODUCTION 

During the past four years an increasing number of articles 
have appeared in the bacteriological literature on the so-called 
“bacteriophage phenomena.” It seems advisable, therefore, 
to give a brief historical review of the data which have accumu¬ 
lated to date and the various theories advanced in regard to this 
subject, before reporting our own experiments. The fundamental 
discoveries will be stated first and then the more recent detailed 
experiments will be discussed as they bear upon the particular 
point under consideration. 

General interest was first attracted to this subject by the work of 
d’Herelle which was published towards the latter part of 1917. It is 
now, however, conceded by the majority of workers in this field, that 
the first observations of this phenomenon were made by Twort in 
1915. 

Twort (1915) was working on the problem of cultivating filtrable 
viruses. In some of his experiments he used glycerinated calf vaccinia. 
He inoculated agar tubes with the vaccinia before the glycerine had 
sterilized it completely, and found that the colonies of yellow and 
white micrococci that grew out, showed what he described as “watery” 
areas. These areas could not be sub-cultured. He found, furthermore, 
that if the water of condensation from these tubes was plated, colonies 
of the micrococci developed which also became transparent. If he 
touched a normal colony of the micrococci with some of this “trans¬ 
parent material,” the normal colony would become “watery.”' The 

1 Submitted in partial fulfillment of the requirements for the degree of Doctor 
of Philosophy, in the Faculty of Pure Science, Columbia University, June, 1922. 

49 


50 


ANN GAYLER KUTTNER 


“transparent material” was still active in high dilution (1:1,000,000), 
and remained so after passage through a Pasteur Chamberland filter. 
All attempts to sub-culture the filtrate proved negative. If the filtrate 
of the transparent material was added to an agar slant which was then 
inoculated with a normal culture of the micrococcus, the organism would 
start to grow, but the growth soon become translucent. The filtrate 
used in higher dilution, produced only a small number of transparent 
spots which appeared at various points of the culture. Twort found 
that the transparent material could be transmitted from one young 
culture to another indefinitely. Pie also observed that these clear 
areas appearing on slant would in time be overgrown, but that the 
transparent material was still active when added to a new, growing 
culture of the micrococci. Thus, Twort, in 1915, stated all the funda¬ 
mental characteristics of this phenomenon which have been confirmed 
in practically every detail in connection with all the different “bacterio¬ 
phages,” active against a wide variety of organisms, that have been dis¬ 
covered since. 

To summarize briefly the characteristics of the “transparent material” 
isolated by Twort from micrococci growing out from calf vaccinia: 

1. It can be separated from the organisms from which it is derived 
by filtration. 

2. It can be transmitted indefinitely from one culture to another. 

3. It is most active against young actively growing organisms. 

4. It has no action on dead organisms. 

5. It cannot be transmitted without the presence of organisms in 
any media. 

6. It is active in high dilution, 1:1,000,000. 

7. It is fairly heat resistant, 60° for one hour being necessary to 
destroy it. 

8. The action is not hastened by anaerobiosis. 

9. It increases in quantity when allowed to act on a culture. 

10. In old cultures the activity of the transparent material is dimin¬ 
ished or impeded. 

11. It acts to a less degree on closely related organisms, such as Gram 
positive cocci isolated from boils. 

12. It does not act on organisms unrelated to the micrococci—such 
as Bad. coli, streptococci, tubercle bacilli, yeasts. 

Twort in concluding his article considers most of the hypotheses as to 
the nature of this substance, that are still under discussion today. 
Twort assumes that if derived from the organism itself, it may be either 


Grift 

MAH 20IS23 


BACTERIOPHAGE PHENOMENA 


51 


a stage in the life history of the organism which is filtrable and will not 
grow on any media and stimulates other cultures to pass to the same 
stage; or it may possibly be an enzyme which is secreted by the bacteria 
and leads to their own destruction, a process during which the amount 
of the enzyme is increased. Twort also discusses the possibility that 
this material might be a filtrable virus which is present as a contamina¬ 
tion. Animal inoculation was absolutely negative, this definitely ruling 
out the vaccinia, and Twort found no very good reason for consider¬ 
ing it a non-pathogenic virus. He concluded that it might best be re¬ 
garded as “an acute infectious disease of micrococci.” 

Twort also mentions a bacillus not related to the Bad. coli group, 
isolated from cases of infantile diarrhea which had the property of 
“dissolving itself.” 

Twort’s work did not attract very much attention, and it 
was not until d’Herelle published a series of papers on what 
is now generally considered the same phenomenon, that Twort’s 
contributions to the subject were seriously studied. 

D’Herelle (1917) was studying the effect of the addition of stool 
filtrates obtained from dysentery cases on the growth of the Shiga 
dysentery bacillus. In his original experiment, he obtained daily 
specimens of feces from a dysentery patient and filtered them, adding 
the filtrate to fresh Shiga cultures. He reported this experiment until 
one day when the patient had reached the convalescent stage, he ob¬ 
served that the turbid Shiga culture had after an interval become clear, 
subsequent to the addition of the last stool filtrate. He next found 
that the addition of a small quantity of this cleared culture to another 
turbid young Shiga culture would dissolve the second culture, and that 
he could repeat this process indefinitely from one dissolved culture to 
another. In one instance, he transmitted the lytic principle originally 
obtained from a stool filtrate, through 935 culture generations, in each 
case adding only a minute quantity of the dissolved culture to the next 
tube, so that the original stool filtrate which started the lysis was soon 
eliminated in the successive dilutions. The dissolved cultures were 
not always completely sterilized. If sub-cultured, a small number of 
colonies would be obtained, the character of which will be described in 
detail below. 

The lytic material isolated by d'Herelle has all the characteris¬ 
tics of Twort’s “transparent material” outlined above. The 


52 


ANN GAYLER KUTTNER 


fact that the stool filtrates and their derivatives were active 
only against living bacteria and could be carried on indefinitely 
from one culture to another convinced d’Herelle that he was 
dealing with an ultramicroscopic filtrable virus which was para¬ 
sitic on bacteria, to which he gave the name “Bacteriophagum 
intestinale .” This name “bacteriophage” has now come into 
general use for phenomena of this type, irrespective of the source 
of the material which first starts the lytic process or the particu¬ 
lar organism against which it acts, and in spite of the fact that 
the theory which the name implies, is not accepted by the ma¬ 
jority of investigators at the present time. 

D’Herelle does not believe that phenomena described by him 
relating to the typhoid-dysentery-coli group are the same as 
those described by Twort, and since repeated by Gratia and 
Callow with staphylococci and vaccine virus. D’Herelle 
states that the two phenomena cannot be the same because of 
the differences in heat resistance. Twort in his original article 
says that his “transparent material” resists a temperature of 
52° for one hour, but is destroyed at 60° for one hour. D’Herelle 
considers that one of the essential criteria of true bacteriophage 
action is the fact that the bacteria which persist after the dis¬ 
solving action of the bacteriophage has taken place, are killed 
at a temperature which does not destroy the bacteriophage. 
For instance, the Shiga bacillus is killed at a temperature of 
60°C. for thirty minutes, whereas the particular bacteriophage 
which d’Herelle tested was only destroyed at 65°C. But, as 
Gratia (1921) has pointed out, the. heat resistance of the bac¬ 
teriophage is not a fixed property and depends upon the particu¬ 
lar culture against which the bacteriophage is tested. Thus, 
in Gratia’s experiment, a bacteriophage heated at 56°C. for 
thirty minutes lost its activity against staphylococcus A, but 
retained it for staphylococcus B, and regained it for strain A. 
Callow working in our laboratory has isolated a bacteriophage 
from vaccine virus active against staphylococcus which is dimin¬ 
ished but not completely destroyed at 75° for thirty minutes. 
Thus, the heat resistance of bacteriophages is a variable property 
which depends not only on methods of testing, but also on the 


BACTERIOPHAGE PHENOMENA 


53 


way the bacteriophage is obtained. Any one who has compared 
the action of a staphylococcus bacteriophage isolated from 
vaccine virus and a dysentery or typhoid bacteriophage cannot 
doubt that the same phenomenon is involved in both cases. Cer¬ 
tain minor differences do exist, but they can probably be attrib¬ 
uted to the biological differences between staphylococci and 
members of the typhoid-colon-dysentery groups, since these 
organisms serve as the indicators upon which we depend for 
evidence of bacteriophage action. 

D’Herelle has recently published a book on “Le Bacterio¬ 
phage^ which contains the results of four years of experimental 
work on this subject. Before going on to the theoretical aspects 
of the subject, it may be well to enumerate a few of the most 
important observations made by him, in addition to the funda¬ 
mental principles which apply to all bacteriophage phenomena 
outlined above, from Twort’s work. 

(1) Filtrates from the stools of typhoid and dysentery convalescents in 
a large percentage of cases contain a lytic principle which has the power 
of dissolving or inhibiting the growth of typhoid or dysentery bacilli, 
and which can be transmitted in series indefinitely. 

(2) The lytic principle isolated from stool filtrates is usually not 
absolutely specific, but is active in most cases against two or more 
members of the dysentery, typhoid or colon group. 

(3) The activity of the lytic agent is often feeble on first isolation 
from the stool filtrates, but rapidly becomes more active when trans¬ 
mitted in series. Not only in subsequent generations, is the lysis more 
rapid and the amount necessary to produce lysis from one tube to the 
next much smaller, but also the range of activity may be extended, so 
that a filtrate which on first isolation is active only against Shiga and Y 
dysentery bacilli may after three or four passages with Shiga bacilli 
show activity against typhoid bacilli as well. It is also possible in 
some instances to make the lytic principle active against organisms 
which are not attacked at first, by successive contacts with these 
bacteria. 

(4) Lysis occurs most readily with young cultures of bacteria which 
are multiplying rapidly. Antiseptics or temperatures which in anyway 
retard the growth of the bacteria, although they do not in anyway lessen 
the activity of the lytic principle itself, interfere with the lytic action. 


54 


ANN GAYLER KUTTNER 


(5) It is impossible to transmit the bacteriophage in series in filtrates 
of broth cultures of susceptible organisms. The products of bacterial 
growth are not sufficient, the living multiplying bacteria are absolutely 
necessary. 

These, briefly, were the most important observations made 
by d’Herelle at the time that we began working on this subject 
in November, 1920. His book which reached this country dur¬ 
ing November, 1921, contains many new facts which will be 
discussed below. D’Herelle in his first article published in 
September, 1917, claimed that the lytic principle isolated by 
him from dysentery stools and called by him bacteriophage, 
was an ultramicroscopic living organism parasitic on bacteria, 
and has maintained this point of view. He considers that the 
bacteriophage is a normal inhabitant of the intestinal tract, 
where it is parasitic on Bad. coli. During the course of intestinal 
disease, the bacteriophage becomes parasitic on the invading 
organism. D’Herelle believes that there exists only one bac¬ 
teriophage which by processes of adaptation is capable of attack¬ 
ing different organisms with which it comes in contact. 

The first investigator to question the living nature of the 
bacteriophage was Kabeshima (1920). He advanced the fol¬ 
lowing reasons for thinking it a ferment. 

(1) An extremely small amount of lytic filtrate is sufficient to dissolve 
a fairly large number of bacteria. 

(2) The lytic filtrate is still active after standing for four years in a 
sealed tube. 

(3) It resists heating (moist) at 70°C. for one hour. 

(4) It resists the action of chloroform, toluene, alcohol, ether, carbolic 
acid, and acetone. 

Kabeshima originated the following method of isolating bacterio¬ 
phage: to 1 volume of lytic filtrate add 3 volumes of acetone. Let the 
mixture stand at room temperature for forty-eight hours, shaking at 
intervals. Evaporate the acetone. A yellowish powder remains. The 
lytic action of this powder according to Kabeshima, is stronger than 
that of the original lytic ferment. 

Kabeshima explained the increase of bacteriophage which occurred 
when the lytic principle was brought in contact with a susceptible 


BACTERIOPHAGE PHENOMENA 


55 


organism in the following way: In the course of disease, a certain gland 
in the digestive tract in an effort to digest the invading bacteria, pro¬ 
duces a catalyst. This catalyst activates a proferment contained in 
the bacteria, liberating a ferment which causes the organisms to autolyze. 
In the next generation this ferment acts as the catalyst and activates 
the proferment in the new bacteria, again causing them to autolyze, 
etc. Kabeshima does not state whether he considers the original 
catalyst and the liberated ferment identical, but their mode of origin is 
so different that this does not appear likely. In any case, the catalyst 
and the ferment have both the power of activating the proferment 
present in bacteria. 

The next most important contribution to the subject was made 
by Bordet and Ciuca in a paper published October, 1920. Bor¬ 
det was the first worker to demonstrate that a lytic principle 
such as that described by d'Herelle could be obtained without 
starting from a stool filtrate. 

By injecting a certain strain of Bad. coli into a guinea-pig intraperi- 
toneally, three or four times at intervals of a few days, Bordet and 
Ciuca obtained an exudate containing a large number of leucocytes 
and a few living bacilli. He added several volumes of broth to this 
filtrate, allowed it to stand at room temperature a few hours or a day 
and then filtered. This filtrate when added to a normal culture of the 
colon bacillus produced lysis transmittable in series. 

D’Herelle in his early experiments described bacteriophages of such 
great activity that they sterilized the cultures completely, and no 
growth was obtained when the dissolved cultures were plated. Bordet 
and Ciuca were the first investigators to show that in most cases bac¬ 
teriophage action did not completely sterilize the culture that had been 
dissolved, but that if such a culture were plated a small number of 
colonies would develop that presented peculiar characteristics. They 
develop more slowly than normal colonies and have a tendency to be 
irregular in outline, and grow discreetly. If these colonies are trans¬ 
planted to broth, the broth never becomes definitely turbid, and the 
supernatant fluid of this broth culture, if added to a normal colon cul¬ 
ture, has lytic power. In other works, certain colonies that grow out 
after lysis has taken place, carry the lytic power. Border and Ciuca 
found that if these cultures were sub-cultured on agar for several genera¬ 
tions, they would grow luxuriantly, and often present a glassy mucoid 
type of growth. They found that these mucoid coli were more virulent 


56 


ANN GAYLER KUTTNER 


and less easily phagocyted than normal Bad. coli. They also reported 
that if a guinea pig received an M.L.D. of Bad. coli and then received an 
injection of dissolved culture heated at 58° for thirty minutes, this 
animal survived, whereas the control died in eight hours. 

Bordet and Ciuca like Kabeshima, did not agree with d’Herelle that 
bacteriophage action was due to a living virus. They interpreted this 
phenomenon as a hereditary tendency for a given culture to autolyze, 
acquired during exposure to unfavorable circumstances, such as are 
produced by the leucocytic response in the peritoneal cavity of the 
guinea pig. 

Bordet and Ciuca (1921 a) were the first workers to produce an anti- 
lytic serum by inoculating rabbits with increasing amounts of dissolved 
culture filtrate. The serum of a rabbit treated in this way, was able, 
according to Bordet and Ciuca, to neutralize the lytic action of their 
bacteriophage completely. If equal quantities of the lytic agent and the 
lytic immune serum were added to a culture of Bad. coli, normal growth 
would be obtained, whereas, if in a parallel tube the lytic agent alone 
was added, no growth occurred. Bordet and Ciuca also showed that 
the antilytic serum could change a lytic irregular colony such as has been 
described above, back to a normal colony in the following way. Two 
agar slants were inoculated, respectively, with 7 drops of the antilytic 
serum and 7 drops of normal rabbit serum, and incubated overnight. 
The following day these two slants and one agar slant to which nothing 
had been added, were inoculated with a lytic colon culture. The slant 
which had been in contact with the antilytic serum showed confluent, 
normal growth after eighteen hours; the other two slants, sparse growth 
of irregular colonies. If these three slants were transplanted to broth, 
the Bad. coli that had grown in the presence of the antilytic serum would 
cloud the broth uniformly, whereas the broth transplants from the other 
two slants would remain clear. Thus, the antilytic serum could inhibit 
or prevent the inheritance of the lytic quality. 

The important contributions made by Bordet and Ciuca are: 

(1) That a lytic agent transmittable in series could be demonstrated 
in the leucocytic exudate obtained by repeated injections of Bad. coli 
into the peritoneal cavity of a guinea pig. 

(2) That after lytic action had taken place on a given culture, certain 
colonies developed on subculture which carried the lytic agent in subse¬ 
quent generations. 

(3) That the lytic agent was antigenic; the antilytic serum was able 
to prevent the action of the lytic agent, and also convert a lytic-bearing 
colony to a normal colony. 


BACTERIOPHAGE phenomena 


57 


Thus, three fundamentally differing theories had been ad¬ 
vanced when we began working on the subject of bacteriophage. 
D’Herelie attributed these phenomena to an ultramicroscopic 
virus parasitic on bacteria, a normal inhabitant of the intestinal 
tract of man and animals; Kabeshima, to a new type of enzyme 
which attacked living cells and was liberated in increased quan¬ 
tity from the substrate on which it acted, and lastly, Bordet 
and Ciuca, to an acquired hereditary tendency to autolyze, 
induced by the action of leucocytes on bacteria. 

I. OBSERVATIONS ON THE ISOLATION OF THE LYTIC AGENT BY 
THE METHODS OF BORDET AND CIUCA, AND OF d’hERELLE 

Bordet and Ciuca’s experiment in which they produced 
Bad. coli bacteriophage experimentally rather than isolating it 
from stool filtrate in the course of a pathological condition, 
seemed of the utmost importance from the point of view of ex¬ 
plaining the nature of the lytic agent. Our first attempt was, 
therefore, to duplicate the result of these workers. 

Ten guinea-pigs were injected with 10 different strains of 
Bad. coli and the exudate treated exactly according to the direc¬ 
tions of Bordet and Ciuca. In no instance, however, was any 
lytic activity of the exudate demonstrable. Wollstein (1921) 
repeated Bordet and Ciuca’s original experiment, and recom¬ 
mended injecting the guinea-pig once instead of three or four 
times. We have been able in 2 guinea-pigs to produce a lytic 
exudate by a single intraperitoneal injection of typhoid bacilli. 

It is the opinion of all workers, however, that this method of 
obtaining the bacteriophage principle is extremely unreliable, 
and that the factors that determine its development are not 
understood. Bail has recently reported that he has been able 
to obtain lytic exudate by the injection of Shiga bacilli, using a 
single dose of such a size that the pig would require two or three 
days to die. Gratia has also used this method successfully with 
staphylococci, but states that he cannot control the various factors 
in the experiment and that he cannot be sure of obtaining a lytic 
exudate every time. 


58 


ANN GAYLER KUTTNER 


D’Herelle has never been able to repeat this experiment of 
Bordet and Ciuca. He considers the development of lytic exu¬ 
dates as due to the permeation of the intestinal wall by a bac¬ 
teriophage which preexisted in the intestine of the pig, as a 
result of the irritation due to infection in the peritoneal cavity. 
He attributes the irregularity of these experiments to the fact 
that the presence of a bacteriophage in the intestine of a particu¬ 
lar guinea-pig active against, or at least capable of adaptation 
against the strain injected, is purely accidental. D’Herelle 
does not include any guinea-pigs in his series investigating the 
presence of bacteriophage in the feces of normal animals. Re¬ 
cently, however, Lisbon'and Carrere (1922) have attempted to 
show that the origin of the bacteriophage is in leucocytes rather 
than in the intestine, but their experiment is not clear cut. They 
produced sterile abscesses in a series of 5 animals by the injec¬ 
tion of sterile petroleum. They were able to isolate a lytic 
agent active against Shiga dysentery bacilli from the pus thus 
obtained, in every case. They state, however, that the feces 
of 4 out of 5 animals tried contained a bacteriophage active against 
Shiga bacilli. The feces of these animals were not examined 
before the abscesses were induced, so that it is difficult, since 
we are dealing with an extremely diffusible substance, to say 
whether or not the bacteriophage originated in the leucocytes 
or in the intestine. Since this method of obtaining a bacterio¬ 
phage principle was first described by Bordet and Ciuca, so 
many simpler methods of starting the lytic process have been 
discovered that give greater promise of solving the nature of 
these phenomena, that it has not seemed worth while to analyze 
the conditions obtained by peritoneal injections more closely. 
The fact that leucocytes may play some role in starting the lytic 
process is also suggested by the fact that staphylococcus bac¬ 
teriophage is so easily isolated from green calf vaccine virus and 
also, as shown by Callow, from boils. 

We may state, however, that while we were unable to obtain 
a lytic exudate by the method of Bordet and Ciuca with 11 
strains of Bad. coli, we succeeded in two instances when typhoid 
bacilli were injected. 


BACTERIOPHAGE PHENOMENA 


59 


Isolation of a typhoid bacteriophage by the d’Herelle technique 

A bacteriophage principle was isolated by the technique dis¬ 
covered by d’Herelle, from the stool of a typhoid convalescent, 
Ida Olsen, sent to me by the courtesy of Dr. Krumwiede of the 
Research Laboratory of the Health Department. 

The stool was plated and typhoid bacilli isolated. Nothing abnormal 
was noted in the appearance of the typhoid colonies. They agglutinated 
readily on the slide in a typhoid immune serum in a dilution of 1:100. 
A small particle of feces was emulsified in broth and incubated over¬ 
night. On the following day twice the volume of broth was added, the 
emulsion was centrifuged, and filtered through a Berkefeld filter. This 
filtrate was tested out in the following way, against the homologous 
typhoid strain, no. 18. 

(a) 2 cc. extract broth + 1 loop of four hour broth culture no. 18 + 
0.5 cc. stool filtrate. 

(b) 2 cc. extract broth + 1 loop of four hour broth culture no. 18 + 
0.5 cc. sterile salt. 

These tubes were incubated for three hours and then left standing 
overnight at room temperature. At the end of this time the control 
tube (b) was definitely cloudy, and the tube with the filtrate (a) still 
clear. One half of the tube (a) was now added to 2 cc. extract broth 
-f* 1 loop of a young broth culture of typhoid no. 18. This second tube 
also failed to show growth as compared with the control on the following 
day. The inhibitory property of tube (a) could be carried on from one 
generation to another indefinitely. It was also found that one of these 
inhibited tubes could dissolve a young typhoid culture which was defi¬ 
nitely turbid, clarifying it completely. The lytic and inhibitory action 
was stronger after a few generations in contact with typhoid bacilli 
than it was in the original stool filtrate. 

This lytic principle which has been isolated for over a year 
and a half at the present writing and with which most of the 
work described in this paper was done, differs in no essential 
from the many bacteriophages described by d’Herelle. The Olsen 
bacteriophage acts non-specifically within the typhoid, colon, 
dysentery group. It is lytic and inhibitory for Shiga, Mt. 
Desert and Flexner dysentery bacilli. In the original experi¬ 
ments when the Olsen bacteriophage was not far removed from 


60 


ANN GAYLER KUTTNER 


the original stool filtrate, it showed no action on paratyphoid 
A or B. Blit when tried again recently against these cultures, 
it was active against both of these organisms. The Olsen bac¬ 
teriophage has no lytic action against laboratory stock strains of 
Bact . coli-communis and communior. It does, however, act 
against a recently isolated Bact. coli which occurred in the stool 
culture of a typhoid carrier referred to below. Typhoid strains 
show the greatest variation in susceptibility to lytic agents. 
The Olsen bacteriophage has acted on most of the typhoid cul¬ 
tures tried, whether a stock culture (Mt. Sinai) or recently iso¬ 
lated strains, with the exception of the Rawlings strain. It 
has recently been possible to obtain a susceptible variant of the 
Rawlings culture (see below). D’Herelle states that from the 
point of view of susceptibility to lytic agent, both the typhoid 
group and the colon group must be regarded as heterologous, 
whereas the members of dysentery groups are all fairly suscep¬ 
tible. The Olsen bacteriophage has no action against a stock 
strain of cholera or against any Gram positive organism that 
has been tried, such as the pneumococcus or staphylococcus. 
No systematic attempts to acclimatize the Olsen bacteriophage 
to organisms against which it was not active have been made. 
The fact that the range of activity of a particular bacteriophage 
could be extended was first shown by d’Herelle and has since 
been repeated by other workers, notably Bordet and Ciuca 
(1921b). This adaptation of the bacteriophage is of theoretical 
importance and must be taken into account in any theory that 
is advanced. 


Types of colonies developing after lysis 

The Olsen bacteriophage never completely sterilized the cul¬ 
tures which it dissolved or inhibited. On subculture it was 
found that a small number of colonies developed which were 
in most cases of two types; one a typical round typhoid colony, 
the other an irregular jagged colony. The latter type if fished 
into broth, produced little or no clouding and the supernatant 
fluid could be shown to contain the lytic agent in the same way 
that Bordet and Ciuca had demonstrated in connection with 


BACTERIOPHAGE PHENOMENA 


61 


the lytic-bearing colonies of Bad. coli. The round colony when 
fished to broth grew normally and the supernatant fluid had no 
lytic action. 

These two types of colonies, one a normal colony, the other 
the lytic carrying colony were obtained with every culture against 
which the Olsen bacteriophage was active, dysentery as well as 
typhoid, and had not been described by d’Herelle at the date of 
our first communication, February 16, 1921 (Kuttner, 1921). 
If one of the normal colonies is restreaked, nothing but typical 
round typhoid colonies develop which apparently do not carry 
the lytic agent. If one of the irregular lytic bearing colonies is 
restreaked, both normal and irregular colonies are obtained. 
In one experiment a lytic colony and a normal colony obtained 
by plating a dissolved culture (Olsen bacteriophage acting on 
the homologous typhoid culture) was restreaked daily for 15 
successive generations. In no case did the restreaking of a normal 
colony yield anything but normal colonies. The restreaking of 
the lytic colonies regularly produced both types: lytic colonies 
and normal colonies. The relative proportion of the two types 
varied, depending upon what part of the lytic colony had been 
touched w r ith the platinum needle. 

Twort in his original article states that the degenerative 
changes he described with micrococci characteristically started 
at the edge of the colony. In working with typhoid and coli 
cultures we have observed a great variation in the lytic colonies 
which appears to be a fairly definite characteristic of certain 
strains. Thus, if the Olsen bacteriophage acts on a Mt. Sinai 
strain of typhoid, the lytic action is most intense at the center 
of the colony. Similarly, with another bacteriophage (Newton) 
acting on a Bad. coli culture, lysis also begins at the center 
and has done so consistently with this strain. In restreaking a 
lytic colony, the greatest proportion of lytic colonies are obtained 
if the original lytic colony is touched at the point where the lytic 
process appears to be most active, whether this happens to be 
at the center of the colony or at the periphery. In our expe¬ 
rience, in most cases, even if the lytic area of the colony was 
touched with platinum wire as delicately as possible, a certain 


62 


ANN GAYLER KUTTNER 


number of what appeared to be absolutely normal colonies 
developed. On the other hand, we do not agree w T ith Bail's 
(1921) statement that after a few generations, the colonies 
carrying the lytic agent revert to normal. This depends as 
pointed out above, upon the manner of restreaking. Bordet 
and Ciuca report that the culture of Bad. coli derived from a 
lytic colony was still lytic after 150 transplants. 

If a series of lytic typhoid or dysentery colonies are ex¬ 
amined under the microscope, it will be found that there are often 
between the lytic colonies, minute transparent granular masses 
that have been called “appearances” by previous workers. 
They are structureless deposits which fail to grow when trans¬ 
planted. It will also be found that the lytic colonies owe their 
irregular shape to the fact that their edges or centers, as the case 
may be, have faded into these “appearances.” All variations 
between a fairly large lytic colony with only a little transparent 
material and the entirely translucent “appearances” which can 
only be seen with the microscope, occur. These transparent 
masses probably represent the debris left when a colony com¬ 
posed entirely of “sensitive” bacilli is dissolved and all the 
organisms killed. 

“ Susceptible ” and “resistant” individuals in a single culture 

Thus it is apparent that the lytic agent separates any sus¬ 
ceptible culture into three types of individuals: The most sus¬ 
ceptible bacillus whose descendents are all represented by the 
“appearance;” the susceptible bacillus whose descendants are 
both sensitive and resistant, represented by the lytic colony; 
and the bacillus which is itself resistant, and gives rise to nothing 
but resistant descendents, represented by the normal colony. 
These three types of individuals must preexist in the culture as 
is indicated in the following experiment: 

A young typhoid culture is washed off in salt solution and the emul¬ 
sion is divided into three tubes and placed in a freezing mixture for 
thirty minutes. In the same way bacteriophage filtrates, undiluted 
and diluted 1:1000 are brought to a temperature of between 5° and 


BACTERIOPHAGE PHENOMENA 


63 


6°C., and just prevented from freezing solid. Some sterile salt solution 
and some centrifuge cups are also cooled. After half an hour, the un¬ 
diluted bacteriophage is added to one tube of emulsion and the diluted 
bacteriophage to another, and the tubes are shaken. The ice salt 
solution is added to the control tube. The mixtures are kept in the 
freezing mixture and plated with the least possible loss of time. In 
both cases with the undiluted and diluted bacteriophage, “appearances,” 
lytic colonies and normal colonies develop, whereas the salt control 
shows only normal colonies. 

This experiment showed that the three types of bacilli pre¬ 
existed in the emulsion. The bacilli in the normal colony had 
been exposed to the same amount of lytic agent as the other 
organisms, and had apparently failed to unite or adsorb any 
bacteriophage, or if they had united or adsorbed the lytic agent, 
were not affected by it. The question that now arose was to 
find out the nature of the union between the susceptible bacilli 
and the lytic agent. Was it a definite union or merely an ad¬ 
sorption? The next step was also carried out at as low a 
temperature as possible, so as to prevent the growth of the bacilli. 

The three tubes of the above experiment were placed in the cooled 
centrifuge tubes, and centrifuged to throw down the organisms. The 
supernatant fluid was poured off, the centrifuge cups and the tubes 
containing the sediment of bacteria recooled. Some of the cold salt 
solution was added and the sediment thoroughly emulsified by means of 
a capillary pipette which had been cooled. This process of washing the 
bacilli was repeated twice, and the emulsion then streaked. 

The tube in which the bacilli had been in contact with con¬ 
centrated bacteriophage yielded “appearances,” lytic colonies 
and normal colonies. The tube to which the diluted bacterio¬ 
phage had been added showed only normal colonies, as did the 
salt control. 

Did the concentrated bacteriophage divide the culture into 
three types of bacilli after one single short contact, or was it a 
question of the concentrated lytic agent not having been com¬ 
pletely removed by the washings? In the latter case it was 
the mechanical adhesion of the bacteriophage to the organisms 


64 


ANN GAYLER KUTTNER 


that had separated the culture into the three individuals of 
different degrees of susceptibility as they grew out in the agar 
streak. But in any case, it is possible to state that all susceptible 
cultures contain individuals of three different potentialities in 
respect to lytic agents. Whether the so-called resistant individ¬ 
uals that make up the normal colony represent, as has been 
claimed, the descendants of an individual that became immune 
to the small amount of bacteriophage that adhered to it, and has 
conferred this immunity to his descendants; or whether the 
physical surface of different bacteria vary so that the lytic agent 
adheres more readily to some individuals than to others, remains 
an open question. 

Bacteriophage action, therefore, seems to depend on two main 
factors: the concentration of the lytic agent and the number 
of susceptible bacteria. This is well illustrated by the action 
of the lytic agent on cultures on solid media. If the lytic agent 
is added undiluted to an agar slant which is subsequently inocu¬ 
lated with a susceptible culture, practically no growth occurs. 
If high dilutions of the lytic agent are added, growth is normal 
except for certain transparent areas that occur at various points 
of the culture. The number and size of these transparent areas 
diminishes with the increasing dilution of the lytic principle. 
D’Herelle considers that each clear spot represents a colony of 
the virus. He believes that the concentration of the lytic agent 
at definite points in this way is characteristic of living things. 
Gratia’s (1921) interpretation of these facts seems equally 
plausible at the present stage of our knowledge: each culture 
is made up of a whole scale of individuals ranging from the 
most susceptible to the most resistant. As the lytic agent is 
diluted, it is capable of dissolving only the most sensitive 
organisms which occur at various points of the culture. 

It seemed of interest to determine whether the normal colonies 
that developed after lysis were totally and permanently refrac¬ 
tive to further exposure to the lytic agent. 

A normal resistant colony obtained by the action of the Olsen bac¬ 
teriophage on strain 18 was isolated and to this culture the lytic agent 


BACTERIOPHAGE PHENOMENA 


65 


was again added in a dilution of 1:10. Lysis occurred more slowly 
than with a normal culture, but was definite. The tube was streaked 
and a resistant colony isolated and this second culture again exposed to 
the lytic agent. This was repeated four times and in the last case, 
definite clearing with the resistant culture did not take place, although 
on streaking the turbid culture a small number of lytic colonies were 
obtained. The resistant culture obtained after four successive expo¬ 
sures to the lytic agent was subcultured for five generations on agar 
and then again exposed to the lytic agent. It has lost a great part of 
its resistance and was nearly as susceptible as the stock culture. 

Bordet and Ciuca (1921b) fished a lytic colony of Bad . coli 
to broth and kept it in the incubator for eight days and for twenty- 
two days at room temperature. At the end of this time they 
reisolated the culture and found it entirely resistant to lysis. 
They do not state, however, whether it again became susceptible 
on subculture. More recently, Eliava and Pozerski (1921) 
reported that they isolated a Shiga culture that had resisted 
lysis. It w^as resistant to bacteriophage action, but after 8 
transplants it became more susceptible again. 

0 

Variations in typhoid strains—can a non-susceptible strain become 
susceptible? 

The proportion of sensitive and resistant bacilli in a given cul¬ 
ture could, therefore, be varied by exposure to a lytic agent. 
A culture which had become definitely resistant could be made 
susceptible again by transplantation. It seemed probable that 
certain strains of typhoid which were naturally resistant to 
lysis might be rendered susceptible by altering the cultural 
conditions under which they were growing. As stated above, 
the Rawlings strain of typhoid had consistently been resistant 
to lysis by the Olsen bacteriophage. This strain was known to 
ferment xylose slowly, whereas all the other strains that were 
susceptible to this particular lytic agent, fermented xylose 
rapidly. 

It seemed, therefore, worthwhile to obtain a variant of Rawl¬ 
ings which fermented xylose rapidly, and see if it was more 
susceptible to lysis than the stock strain. 


66 


ANN GAYLER KUTTNER 


The Rawlings strain was streaked out on agar plates containing 1 
per cent xylose according to the method described by Morishima for 
obtaining daughter colonies. The plate was sealed with plasticene 
and incubated four days. At the end of this time typical daughter 
colonies had developed which when fished to xylose broth, fermented this 
sugar in twenty-four hours, whereas the stock strain usually required 
seven days to ferment. The xylose rapid fermenting variant of Rawlings 
and the slow fermenting stock culture were now exposed to the Olsen 
bacteriophage, but no lysis was obtained and no lytic colonies developed 
on subculture. 

Since Rawlings was a very old stock culture, it was thought 
possible that it had become resistant, due to the fact that it 
had been held as a stock culture and transplanted at long in¬ 
tervals from dried cultures. It was, therefore, transplanted 
daily for eight generations on moderately alkaline (pH 7.6) 
and very alkaline agar (pH 9); since it had been shown by 
Gratia that an alkaline reaction favors lysis, it was thought that 
possibly susceptible bacilli might develop as the result of growth 
in alkaline media. This did not prove to be the case and no 
evidence of lysis was obtained when these cultures were exposed 
to the Olsen bacteriophage. We have now, however, succeeded 
in obtaining a susceptible variant of the Rawlings strain. At 
the same time that the above experiments were being carried 
out, the stock Rawlings strain was transplanted to 100 cc. broth 
and allowed to remain in the incubator for four months and 
then left at room temperature for two months. The organism 
remained in the broth from June, 1921, until November, 1921. 
It was then isolated and identified as typhoid by fishing to Russell 
double sugar medium, on which it gave a typical reaction, and 
by the agglutination test. This strain reisolated from the broth 
proved susceptible to the Olsen bacteriophage and subsequent 
transplants from this culture have remained so for a period of 
three months. 

Isolation of a resistant variant from an originally susceptible strain 
without exposure to bacteriophage 

In April, 1921, Gratia reported that he had obtained a resistant 
variant from his susceptible Bad. coli culture by reisolating it 


BACTERIOPHAGE PHENOMENA 


67 


from the pellicle formed in an old broth culture. Pinhead, 
glassy colonies protruded from this pellicle, and by subculturing 
one of these, he obtained a resistant culture. The resistant cul¬ 
ture was more motile and also more virulent than the stock cul¬ 
ture. The resistance of this resistant variant was not, however, 
absolute. It was much more marked in acid than in alkaline 
media. 

During May, 1921, it was suddenly noticed that the stock strain 
of typhoid, no. 18, which was transplanted almost daily, had 
become resistant to various agents (see below) which had formerly 
caused it to undergo lysis. It was then tested against the Olsen 
bacteriophage and it was found that no lysis was obtained with 
any of the transplants of no. 18 made after May 14. By going 
back to a culture of April 23, a susceptible transplant of no. 18 
was again obtained. The sudden development of resistance on 
the part of this strain cannot be attributed to aging in this case, 
since the culture was in daily use. It was found that the stock 
agar we had been using for transplanting this culture had been 
somewhat more acid (pH 7) than we had used before, and we 
thought that perhaps the predominance of the resistant individ¬ 
uals over the susceptible might be attributed to this. 

We, therefore, transplanted a susceptible and a resistant variant of 
no. 18 twice daily on acid, pH 6.8, agar and on alkalin pH 8.2 agar for 
twelve generations. We transplanted as rapidly as possible because 
we knew that the maximum number of susceptible individuals occurred 
in young cultures. We then tested these four different cultures (18 
susceptible, twelfth generation, agar pH 6.8; 18 susceptible, twelfth 
generation, agar pH 8.2; 18 resistant, twelfth generation, agar pH 6.8; 
18 resistant, twelfth generation, agar pH 8.2) against the Olsen 
bacteriophage. 

The susceptible strain was still susceptible from both the acid 
and the alkaline medium, and the resistant strain still resistant 
from both media. At the same time, June, 1921, that the above 
experiment was done, the two variants of no. 18 were inoculated 
into 100 cc. of broth each, and allowed to age in the same way 
as the Rawlings culture described above. 


68 


ANN GAYLER KUTTNER 


In summing up our results with various strains of typhoid, 
we may state that we do not understand the factors that determine 
the susceptibility or resistance of a culture. The reaction of 
the medium and the rapidity of transplantation do not seem to 
affect the relation of susceptible and resistant individuals in a 
given culture. A culture that had been isolated for six months 
suddenly became resistant without being exposed to bacterio¬ 
phage, and a different transplant of the same original culture 
has remained susceptible for a year and a half, and has been used 
constantly in various tests. In the case of the Rawlings culture, 
originally resistant, a susceptible variant has been obtained by 
reisolating the strain from an old broth culture. In conclusion, 
we may say that in typhoid cultures the relation between suscep¬ 
tible and resistant organisms is not a very stable one, and that 
for reasons of which we are ignorant, one of the other type may 
predominate. 

II. ISOLATION OF THE LYTIC AGENT FROM THE TISSUES AND BLOOD 
OF NORMAL ANIMALS 

The action of the Olsen bacteriophage on typhoid and dysentery 
cultures was studied in detail in order to familiarize ourselves 
with the lytic process. It was fairly obvious, however, that un¬ 
less one was willing to accept d’Herelle’s theory of a living virus, 
no progress in finding out the nature of the phenomena could be 
made, unless it was possible to isolate a lytic principle without the 
interaction of the living animal body. The fact that the lytic 
agent could be transmitted indefinitely in series and was active 
only against vigorously growing bacilli, suggested that the 
bacteriophage principle might be derived from the organisms 
themselves. 

We proceeded on a theory first advanced by d’Herelle, but 
discarded by him in favor of the living virus. According to this 
theory, bacteriophage action was due either to the activation 
of the natural autolysin contained in all bacteria, or to the re¬ 
moval of an autolysin-inhibiting substance. Once this natural 
autolysin was liberated it could in turn liberate more of the autol- 


BACTERIOPHAGE PHENOMENA 


69 


ysin from the next generation of bacteria and so on indefinitely. 
This hypothesis will be again discussed at the end of the paper. 

The work of Cantacuzene and Marie (1919) on the action of 
intestinal mucosa on cholera vibrioes and that of Turro (1921) 
on the action of tissue extracts on a variety of bacteria, suggested 
that a bacteriophage principle might have played some part in 
the experiments reported by these workers. 

With the work of Cantacuzene and Marie, and Turro in mind, 
we prepared various tissue extracts as has been already reported 
in a brief note. 

Bacteriophage action started with extracts of intestinal mucosa 

Experiment. March 18 the small intestine, large intestine, and a 
part of the abdominal wall of 3 normal guinea-pigs which had been bled 
to death for complement, were removed. The tissues were thoroughly 
washed in running water and cut into small pieces with a scissors. With¬ 
out any preliminary drying each of the three types of tissue was divided 
roughly into 4 parts and placed in 12 bottles. Each type of tissue was 
extracted in 50 per cent glycerol, 25 per cent glycerol, 5 per cent glycerol 
and salt solution, 50 cc. of fluid being added to each bottle. The 
bottles were put on ice from March 18 until March 21 and then plated 
to see to what extent they were contaminated. The tissue to which the 
50 per cent glycerol had been added showed no growth. The plates 
from the other bottles developed a small number of colonies, mainly 
staphylococci. 

All the bottles were then incubated from March 22 to March 29. 
On March 29 a small amount of the supernatant fluid from the bottles of 
small intestine, large intestine and abdominal wall extracting in 50 per 
cent glycerol were centrifuged and filtered through a Berkefeld filter 
to obtain a clear fluid. These filtrates were titrated to make sure that 
the reaction was neither sufficiently acid nor alkaline to interfere with 
the growth of the typhoid bacillus. The filtrates were tested in the 
following way against the typhoid strain 18, 0.1 cc. of a fairly heavy 
saline emulsion from one eighteen hour agar slant being added to 2 
cc. extract broth, pH 7.8. The amount of emulsion added was sufficient 
to make the tubes slightly but definitely turbid. 


70 


ANN GAYLER KUTTNER 


March 80 


TUBE 

AMOUNT 

OP 

BROTH 

CUL¬ 

TURE 

18 

PREPARATIONS TESTED 

4 

HOURS 

24 

HOURS 


1 

CC. 

2 

CC. 

0.5 

0.5 cc. 50 per cent glycerol, 

+ + 

+ 1 

Normal growth 

2 

2 

0.5 

small intestine, pH 6.8 
0.25 cc. 50 per cent glycerol, 

+ + 

+ 

Few lytic colo¬ 

3 

2 

0.5 

small intestine, pH 6.8 

0.50 cc. 50 per cent glycerol, 

+ + 

+ + 

nies 

Normal growth 

4 

2 

0.5 

large intestine, pH 7.0 
0.25 cc. 50 per cent glycerol, 

+ + 

+ + 

Normal growth 

5 

2 

0.5 

large intestine, pH 7.0 

0.50 cc. 50 per cent glycerol, 

+ + 

+ 

Normal growth 

6 

2 

0.5 

abdominal wall, pH 6.8 
0.25 cc. 50 per cent glycerol, 

+ + 

+ 1 

Normal growth 

7 

2 

0.5 

abdominal wall, pH 6.8 
0.50 cc. 50 per cent glycerol, 

+ + 

+ + 

Normal growth 

8 

2 

0.5 

solution 

0.25 cc. 50 per cent glycerol, 

+ + 

+ + 

Normal growth 

9 

2 

0.5 

solution 

0.50 cc., Olsen bacterio¬ 

d= 


Normal growth 

10 

2 

0.5 

phage 

0.25 cc., Olsen bacterio¬ 

=fc 

dfc 

Lytic colonies 

11 

2 

0.5 

phage 

0.50 cc., salt solution 

+ + 

+ + 

Normal growth 

12 

2 

0.5 

0.25 cc., salt solution 

+ + 

+ + 

Normal growth 


+4* = turbidity equal to control. 
— = complete clearing. 


The tubes were placed in the incubator for four hours and examined, 
and then put back in the incubator for twenty hours more, and again 
examined for clearing. The tubes were all plated at this time and then 
heated at 56°C. for thirty minutes to prevent the overgrowth of resistant 
types. 

The twenty-four hour plate of tube 2 showed lytic colonies identical 
with those of tube 10, the lytic control tube. The platings from none 
of the other tubes showed any lytic colonies. Tube 1 where a greater 
amount of active intestinal extract was used showed no lytic colonies, 
but this is often the case, since tubes to which a greater amount of the 
lytic agent whatever its origin is added, usually clear faster and are then 
often more rapidly overgrown with the resistant organisms. The same 
thing occurred in this particular experiment in the control tube 9. 
Glycerol alone failed to give rise to lytic colonies. The degree of clear¬ 
ing is a very unreliable criterion, especially on first isolation of a lytic 












BACTERIOPHAGE PHENOMENA 


71 


agent. The development of lytic colonies is the most definite proof 
of the presence of a lytic agent. Clearing alone is never sufficient 
evidence for bacteriophage action. 

The lytic colonies obtained from tube 2 were studied in detail and 
found to have all the characteristics of lytic colonies obtained by action 
of the Olsen bacteriophage. 

April (1) The supernatant fluid of the bottle with the small intestine 
extracting in 25 per cent glycerol was centrifuged and filtered through a 
Berkefeld. Another lot of the bottle with small intestine and 50 per cent 
glycerol was filtered, and then these filtrates small intestine 25 per 
cent glycerol incubating March 22 to April 1, small intestine 50 per 
cent glycerol incubating March 22 to April 1, together with the first 
extract found active small intestine March 22 to March 29 (tested 
March 30) were set up on April 1. Tube 2 in the previous protocol 
was also tested to see whether the lytic agent isolated from the mucosa 
was transmissible in series. The test was set up in the same number as 
the previous one. 

April 1 


TUBE 

AMOUNT 

OF 

BROTH 

CUL¬ 

TURE 

18 

PREPARATIONS TESTED 

PLATES AFTER 
TWENTY-FOUR HOURS 


CC. 

CC. 



1 

2 

0.5 

0.50 cc. 50 per cent glycerol, small intes¬ 

Normal growth 




tine, March 22 to March 29 


2 

2 

0.5 

0.25 cc. 50 per cent glycerol, small intes¬ 

Many lytic colo¬ 




tine, March 22 to March 29 

nies 

3 

2 

0.5 

0.50 cc. 50 per cent glycerol, small intes¬ 

Normal growth 




tine, March 22 to April 1 


4 

2 

0.5 

0.25 cc. 50 per cent glycerol, small intes¬ 

Many lytic colo¬ 




tine, March 22 to April 1 

nies 

5 

2 

0.5 

0.50 cc. 25 per cent glycerol, small intes¬ 

Normal growth 




tine, March 22 to April 1 


6 

2 

0.5 

0.25 cc. 25 per cent glycerol, small intes¬ 

Many lytic colo¬ 




tine, March 22 to April 1 

nies 

7 

2 

0.5 

0.50 cc. tube 2, March 30 

Many lytic colo¬ 





nies 

8 

2 

0.5 

0.25 cc. tube 2, March 30 

Many lytic colo¬ 





nies 

9 

2 

0.5 

0.50 cc. Olsen bacteriophage 

Many lytic colo¬ 





nies 

10 

2 

0.5 

0.50 cc. salt 

Normal growth 


JOURNAL OF BACTERIOLOGY, VOL. VIII, NO. 










72 


ANN GAYLER KUTTNER 


The 25 per cent glycerol extraction of the pooled small intestine 
tissue was also found active; the result obtained with the 50 per cent 
glycerol on March 30 was repeated. The lytic agent derived from 
the mucosa was found to be active in the second generation (tubes 7 
and 8). 

The lytic process started with these extracts in no way differed from 
the bacteriophage action started with Olsen bacteriophage. It could be 
transmitted in series and, the speed of lysis and number of lytic colonies 
obtained increased markedly in the first three or four generations. 
Extracts of the large intestine with 50 per cent glycerol also proved 
active after a longer period of extraction March 22 to April 25. Extracts 
of the abdominal wall were never found to be lytic. The preparations 
with 50 per cent glycerol and salt in spite of the addition of 5 per cent 
chloroform, became so contaminated that they were discarded without 
being tested. 

It was thus shown that lysis transmittable in series could be 
started with glycerol extracts of large and small guinea-pig 
intestinal mucosa in certain instances. We also tried to see if 
this property was confined to the intestinal mucous membrane, 
or whether it could also be found in other organs. We, there¬ 
fore, prepared a liver extract exactly according to the directions 
of Turro (1921). 

Bacteriophage action started with an extract of liver tissue 

March 31, 1921, a liver taken from a normal guinea pig was minced, 
shaken up in about 6 volumes of acetone, dried in vacuo and pulverized. 
To approximately 1 gram of liver powder, 20 cc. of sterile salt solution 
were added. To one tube 40 drops of chloroform were added, to the 
other a small amount of dry sodium fluoride. Both tubes were incu¬ 
bated fifteen hours. At the end of this time the tube to which the 
sodium fluoride had been added was contaminated with a large Gram 
positive bacillus. The tube to which the chloroform had been added 
was apparently sterile. The former was centrifuged and filtered 
through a Berkefeld, the other was merely centrifuged. Both these 
preparations were tested against typhoid no. 18 in the same way as the 
intestinal extracts. The liver extract to which the chloroform had 
been added was not as active in the first generation as the one to which 
sodium fluoride had been added. The following protocol where the 
specificity of the liver extract is tested is of interest. 


BACTERIOPHAGE PHENOMENA 


73 


The liver extract (sodium fluoride March 31) was tested against the 
following 6 strains: typhoid no. 18, typhoid Rawlings, Shiga dysentery, 
Mt. Desert, cholera and the Bad. coli obtained from Dr. Bordet. In 
this case, the extract was tested against each culture in two ways by 
lysis and by inhibition; that is, in the former case the 2 cc. of broth, 
the amount used consistently throughout these tests, was inoculated 
with a sufficient amount of the culture emulsion taken from an eighteen 
hours slant to produce definite clouding. In the latter case the tube 
was merely heavily inoculated without producing visible turbidity. 
This method of setting up the tests in two ways is to be recommended 
where a new and feeble lytic agent is being tested, because the danger of 
overgrowth with resistant types always exists. By inoculating a large 
and small number of organisms, the chances of finding lytic colonies or 
transparent areas, which is the surest indication of bacteriophage ac¬ 
tion, at one or the other intervals of plating, is increased. In the major¬ 
ity of our more recent tests we have adopted the following routine: The 
tubes are plated after two hours and then left standing at room tempera¬ 
ture, and then plated again. 


April 12, 1921 


TUBE 

AMOUNT 

OF 

BROTH 

CULTURE 

LIVER 

(NaFl) 

EX¬ 
TRACT 
APRIL 1 

PLATED AFTER FOUR HOURS 


CC. 



CC. 


1 

2 

Typhoid no. 18 

0.50 cc. 

0.2 

Few lytic colonies 

2 

2 

Typhoid 

0.05 cc. 

0.2 

Little growth, few lytic colo¬ 






nies 

3 

2 

Rawlings 

0.50 cc. 

0.2 

Normal growth 

4 

2 

Rawlings 

0.05 cc. 

0.2 

Normal growth 

5 

2 

Shiga 

0.50 cc. 

0.2 

Transparent areas 

6 

2 

Shiga 

0.05 cc. 

0.2 

Very delicate growth, no lytic 






colonies 

7 

2 

Mt. Desert 

0.50 cc. 

0.2 

Normal growth 

8 

2 

Mt. Desert 

0.05 cc. 

0.2 

Transparent areas 

9 

2 

Cholera 

0.50 cc. 

0.2 

Normal growth 

10 

2 

Cholera 

0.05 cc. 

0.2 

Normal growth 

11 

2 

Bordet B. coli 


0.2 

Normal growth 

12 

2 

Bordet B. coli 


0.2 

Normal growth 












74 


ANN GAYLER KUTTNER 


A similar series of tubes was set up at the same time in which sterile 
salt solution was substituted for the liver extract. The plates from 
these tubes all showed normal growth. The action of sodium fluoride 
alone was also tried, and in those concentrations which did not interfere 
with growth, normal colonies were obtained. It is interesting to note 
that the Rawling strain is resistant to the liver extract in the same way 
that it is to the Olsen bacteriophage. The range of activity of this 
liver extract was about the same as that of the Olsen bacteriophage. 
Cholera and the Bordet Bad. coli were not affected by either. 

The lytic process started with the liver extract was shown to 
be transmissible in series. In order to demonstrate even more 
clearly that the same substance was at work in the liver extract 
and in the Olsen bacteriophage, 2 rabbits were immunized, one 
with dissolved typhoid culture filtrate (Olsen bacteriophage), 
the other with the active liver extract. The rabbits were in¬ 
jected intravenously at intervals of four or five days. The rabbit 
1098 injected with the Olsen dissolved culture filtrate, was bled 
the 4th day after the sixth injection. The rabbit 335 injected 
with active liver extract, was bled one day after the fourth 
injection. 

The normal and immune sera of these two rabbits were then 
tested as to their ability to convert lytic colonies to normal by 
the method described by Bordet (1921c). 

Experiment. June 13, 1921, 4 agar slants were inoculated in the 
following way: 

(1) with 7 drops of the normal serum of rabbit 1098. 

(2) with 7 drops of the immune serum of rabbit 1098. 

(3) with 7 drops of the normal serum of rabbit 335. 

(4) with 7 drops of the immune serum of rabbit 335. 

These tubes were then incubated in the inclined position overnight. 
On the following day they were found to be sterile. They were then 
inoculated with equal amounts of a broth fishing of a lytic colony ob¬ 
tained by the action of the Olsen bacteriophage on no. 18. 

(5) A plain agar slant was also inoculated with the same material. 
After eighteen hours the following result was obtained: 

Tube 1 showed a small number of lytic and normal colonies. 

Tube 2 showed heavy confluent growth. 


BACTERIOPHAGE PHENOMENA 


75 


Tube 3 showed a small number of lytic and normal colonies. 

Tube 4 showed a fair amount of confluent growth, no lytic colonies. 

Tube 5 showed a small number of lytic and normal colonies. 

This experiment was repeated two days later, and the same result was 
obtained. The antilytic serum produced by injecting the active 
extract (liver) had the same power to neutralize the growth of lytic 
colonies as the antilytic serum produced by the inoculation of Olsen 
bacteriophage. The normal serum of neither of the rabbits interfered 
with the development of the lytic colonies. 

Bacteriophage action started with normal rabbit serum 

Bordet in describing the production of antilytic sera, mentioned 
the fact that the lytic agent persisted in the circulation for a 
period of forty-eight hours. In starting to immunize 2 rabbits, 
one with Olsen dissolved culture filtrate no. 1052, the other with 
active liver extract no. 335, we took normal bleedings and also 
bled again eighteen hours after the first injection to see whether 
the lytic agent could still be demonstrated in the circulation. 
We then tested the four bleedings obtained in this way as follows: 


TUBE 

AMOUNT 

OP 

BROTH 

CUL¬ 

TURE 

18 

SERA TESTED 

SIX HOUR PLATES 


CC. 

CC. 



1 

2 

0.3 

0.2 cc. normal rabbit serum no. 335 

No lytic colonies 

2 

2 

0.3 

0.2 cc. rabbit serum no. 335 after 

Some lytic colonies and 




first injection 

transparent areas 

3 

2 

0.3 

0.2 cc. normal rabbit serum no. 

Some lytic colonies 




1052 


4 

2 

0.3 

0.2 cc. rabbit serum no. 1052 after 

No lytic colonies 

5 

2 

0.3 

first injection 

0.2 cc. salt solution 

No lytic colonies 


It was thus found that normal rabbit serum in some instances 
contained a lytic agent. Transmission of the lytic agent in 
series from tube 3 offered no difficulties. This experiment was 
repeated three times. The suspicion that the two tubes of no. 
1052 had been accidentally mixed could be definitely eliminated 
because, in taking the normal bleeding, a large quantity of blood 
had been taken, since the normal serum would be required as 











76 


ANN GAYLEB KUTTNER 


a control for future tests, whereas, only a small amount of blood 
was necessary for the demonstration of the persistence of the 
lytic agent in the circulation. It is also to be noted that the 
serum of the rabbit 335 injected with one dose of active liver 
extract, was active after eighteen hours. 

During the past year out of 50 normal rabbits bled, the serum 
of 6 contained a lytic agent active against typhoid bacilli. No 
data were obtained as to what determined the presence of the 
bacteriophage principle in the circulation of these particular 
animals. The serum of 2 of these rabbits when bled again, 
failed to show any lytic activity, indicating that the condition 
was transitory. An active serum remained so for an indefinite 
period when stored on ice. The lytic agent in the serum was 
not related to the complement since the activity was not de¬ 
creased by inactivation at 56° for thirty minutes, and in two 
instances seemed definitely increased after inactivation. The 
activity of two immune sera, one rabbit and one horse, was 
also tested, but they were found negative. 

Possible connection between bactericidal titer of normal rabbit sera 
and bacteriophage action 

It is of interest in this connection to recall the observations 
of Teague and McWilliams (1917) who found a great variation 
in the bactericidal titer of different rabbit sera, and observed that 
the bactericidal titer of immune rabbit serum was much lower 
than that of normal rabbit serum. It seemed possible that the 
explanation of the exceptionally high bactericidal titer of certain 
rabbits might be attributed to bacteriophage action, but had 
this been the case, it would have been extraordinary if the ex¬ 
tremely characteristic lytic colonies had not been observed 
before. We consequently duplicated as closely as possible the 
tests made by Teague and McWilliams with one of our lytic 
sera. We were short of active serum and could use 0.7 cc. only 
instead of 1 cc. Seven tenths cubic centimeter of serum was 
inoculated with a fairly light suspension of typhoid bacilli and 
plated after five hours incubation and again after eighteen hours. 


BACTEKIOPHAGE PHENOMENA 


77 


In both five-hour and eighteen-hour plates a small number of 
lytic colonies were obtained. We did not count the typhoid 
suspension accurately, and it is quite possible that with the use 
of such a large amount of serum and inoculating a relatively small 
dose of bacteria, only the resistant colonies might develop, giving 
a small number of perfectly typical colonies. Teague and Mc¬ 
Williams used streak plates in the same way that we do in de¬ 
termining lytic action. It is customary in most bactericidal 
tests to make “pour” plates, in which the recognition of a small 
number of lytic colonies would be extremely difficult. Whether 
or not bacteriophage action played a part in the results obtained 
by previous workers cannot be definitely stated, but in the future 
any one working on the bactericidal titer of normal rabbit sera 
will have to test for bacteriophage action. The differentiation 
of bactericidal action and lytic action can easily be made because 
of the heat resistance of the lytic agent. A small number of 
normal guinea-pig sera were also tested against typhoid, but 
none of them showed any lytic activity. A few isolated experi¬ 
ments with normal human sera were negative. 

The lytic agents found in serum, like those described from 
other sources, were resistant to drying. One of the active sera 
which had dried completely in the bottom of a tube, when re¬ 
dissolved in a little salt solution, still proved active. We found 
that we were unable to absorb out the lytic agent from a serum 
by a single absorption with typhoid bacilli at 0°C. 

Absorption of lytic agent from serum 

One cubic centimeter of active rabbit serum no. 1052 and the saline 
emulsion of a fresh agar slant of the Mt. Sinai typhoid strain were both 
placed in a freezing mixture for thirty minutes. The serum and the 
bacteria were then combined and kept at a freezing temperature for 
four hours. The experiment was carried out at the low temperature to 
prevent the growth of the bacteria since we know that the amount of the 
lytic agent is increased with the growth of the organisms. At the end of 
this time, the mixture was centrifuged to throw down the organisms. 
The serum was pipetted off and heated at 58° for thirty minutes to kill 
any remaining bacteria. In order to prove that some of the lytic agent 


78 


ANN GAYLER KUTTNER 


had actually united with the bacteria, the organisms were washed three 
times (to eliminate the serum which had merely adhered) and then 
streaked out on an agar plate. At the same time, 1 cc. of sterile broth 
was added and the organisms incubated overnight, to see if the growth 
thus obtained would be normal. On the following day, this tube was 
also plated. 

The plate made immediately after washing the bacteria showed no 
lytic colonies, but the plate from the broth tube showed definite evidence 
of lysis. The serum after being tested for sterility, was set up with the 
Mt. Sinai culture to see if the lytic agent had been absorbed out. Typi¬ 
cal lytic colonies were obtained after the serum and culture had been in 
contact for three hours. Thus, four hours absorption was not sufficient 
to remove the lytic agent from the active normal rabbit serum. 

The specificity of one of the active sera was tried out in one 
experiment. We were always very much handicapped by not 
being able to get a very large quantity of active normal serum, 
since we had no way of judging when a serum would be active. 
In this instance, the serum which was lytic for typhoid strain 
no. 18, was not active for Mt. Desert, but too little work has 
been done on this point to draw any conclusions. 

Is the presence of the lytic agent in the circulation secondary to its 
presence in the intestinal canal f 

At the same time that most of these experiments were carried 
out, d’Herelle’s findings on the occurrence of bacteriophage in 
the feces of various normal animals were not known to us, so 
that no attempt was made to correlate the active rabbit sera 
with the presence of bacteriophage in the feces. In his book, 
d’Herelle states that he examined the feces of 2 rabbits and found 
that the filtrate obtained was active in one case against Shiga, 
Mt. Desert and Flexner dysentery bacilli, while the filtrate from 
the other rabbit showed only a very slight activity against the 
Shiga bacillus. When we first discovered the lytic agent in 
normal rabbit sera, we thought that the lytic activity could be 
attributed to some ferment-like activity which activitated the 
bacteria to autolyze, since normal rabbits do not in any way come 
in contact with members of the typhoid-dysentery group. 


BACTEKIOPHAGE PHENOMENA 


79 


But if a Shiga bacteriophage can be isolated from the feces of a 
normal rabbit, it may be argued that the sera of certain rabbits 
which happen to contain a bacteriophage against typhoid or 
Shiga in their intestinal tract, may be active because under certain 
circumstances the bacteriophage penetrates into the circulation 
from the intestine. Since normal rabbits do not have either 
typhoid or Shiga bacilli in their feces, it may be assumed that 
feces of these animals must contain organisms closely related 
to these bacteria in their fecal flora, since the bacteriophage is 
only known to exist in the presence of susceptible bacilli. 

We devised the following experiment in our attempt to obtain 
some information on this point. 

We bled 6 normal rabbits, and at the same time plated the feces of 
each on Endo plates. The fecal flora of rabbits does not seem to be 
very varied, since, in most cases, only two types of organisms were 
obtained, and in some, only one type. The plates were examined care¬ 
fully under the microscope to see if any small lytic colonies of or¬ 
ganisms more or less completely dissolved, could be detected in the 
direct plates, but none were found. The organisms obtained were 
isolated on Russell's medium. The fecal emulsion was incubated 
overnight and then heated at 60° for one hour, since we planned to test 
the fecal suspension for evidence of bacteriophage if any of the sera 
proved active. The sera were tested against the homologous strains 
or strain of Bad. coli, and also against other organisms. The protocol 
of only one rabbit serum will be given to show the method, since the 
experiment was entirely negative. 


Rabbit serum no. 530 inactivated 56° for thirty minutes 


TUBE 

AMOUNT 

OP 

BROTH 

CULTURE 

SERUM 

TWO HOUR 
PLATES 

EIGHTEEN 
HOUR PLATES 


CC. 


CC. 



1 

2 

No. 530 homologous Bad. coli (1) 

0.3 



2 

2 

No. 530 homologous Bad. coli (2) 

0.3 



3 

4 

5 

2 

2 

2 

Typhoid no. 18 

Typhoid Mt. Sinai 

Typhoid Mallon 

0.3 

0.3 

0.3 

No lytic 
colonies 

No lytic 

colonies 

6 

2 

Mt. Desert 

0.3 



7 

2 

Newton Bad. coli 

0.3 















80 


ANN GAYLER KUTTNER 


The sera were thus tried against 7 different strains, 2 of the 
organisms being derived from the same rabbit’s intestinal tract, 
but none of them proved active against either the homologous 
Bad. coli or the other cultures tried. Most of the workers who 
have studied stool filtrates from normal as well as pathological 
human cases, state that bacteriophage action could probably be 
demonstrated in every stool if it were tested against the right 
organisms. But the problem of finding the right organisms is 
not an easy one. This line of research has not been pursued 
further because of the discovery made by several different workers, 
including ourselves, that the bacteriophage principle can be 
obtained from the bacteria alone under certain circumstances 
without the action of any external agent. 

We have thus been able to start lytic action with 2 different 
types of tissues derived from guinea-pigs, and also with normal 
rabbit sera. The intestinal mucosa of certain guinea-pigs ex¬ 
tracted with glycerol when added to normal typhoid cultures 
produced lysis transmittable in series. In the same way liver 
extracts from guinea-pigs can in certain instances produce the 
the same result. The presence of lytic agents in these tissues 
active against typhoid bacilli is, however, extremely rare. Nor¬ 
mal rabbit sera (6 out of 50) also occasionally contain the lytic 
agent for typhoid bacilli, but we do not understand the condi¬ 
tions that determine the lytic activity of the blood of these 
animals. The experiments in which the bacteriophage action 
of these normal tissues or normal sera was shown, were always 
carefully controlled and the culture without the addition of the 
particular extract or serum, never gave any evidence of lysis. 

III. EXPERIMENTS ON THE ORIGIN OF THE LYTIC AGENT IN THE 
BACTERIA THEMSELVES 

Bacteriophage action may thus be started by a wide variety 
of agents from normal as well as from diseased animals, but what¬ 
ever agent starts the process, it is clear that eventually in a series, 
the lytic agent must be derived from the bacteria themselves, 
unless we are willing to accept the parasitic nature of the lytic 
agent. 


BACTERIOPHAGE PHENOMENA 


81 


If the lytic agent is really an activated autolysin, it ought 
to be possible to isolate it from old, spontaneously autolyzing 
cultures. 

On June 13, 1921, two bottles of 100 cc. of broth were inoculated 
respectively with the strain of the stock typhoid strain of typhoid no. 
18 which had spontaneously become resistant to bacteriophage action 
and with the susceptible variant of no. 18. These broth cultures had 
remained in the incubator for a period of four months and had then 
been left at room temperature for two more months. On November 9, 
some of the supernatant fluid from these two bottles was removed, cen¬ 
trifuged and heated at 58°C. for thirty minutes. These heated superna¬ 
tants were then tested against the susceptible variant of no. 18, which 
had been in constant use as a test culture. Large amounts were added 
because it was thought that if present at all, the lytic agent would be 
feeble. The test was set up as follows: 1 cc. amounts of the heated 
supernatants were inoculated directly with 0.1 cc. of no. 18, and at the 
same time, the test was carried out in the usual way, 2 cc. of broth 
being inoculated with 0.1 cc. of culture, and then 0.5 cc. of the material 
to be tested, added. 


TUBE 

AMOUNT 

OP 

BROTH 

CUL¬ 

TURE 

18 

OLD BROTH CULTURES TESTED 

FIVE HOUR PLATES 

1 

CC. 

2 

CC. 

0.1 

1.0 cc. old broth 18D (resistant variant) 

Transparent areas 

2 


0.1 

0.5 cc. old broth 18D (resistant variant) 

Lytic colonies 

3 

2 

0.1 

1.0 cc. old broth 18 (susceptible variant) 

Normal growth 

4 


0.1 

0.5 cc. old broth 18 (susceptible variant) 

Normal growth 

5 

2 

0.1 

1.0 cc. sterile broth 

Normal growth 

6 


0.1 

0.5 cc. salt 

Normal growth 


This test was repeated using the filtrate of this old broth cul¬ 
ture, together with the previously used heated supernatant fluid. 
The same result was obtained. The strain of typhoid was then 
reisolated from this culture. Only normal colonies were ob¬ 
tained on streaking. A fresh agar slant made by transplanting 
one of these colonies was tested against the Olsen bacteriophage. 
It was still wholly resistant. 










82 


ANN GAYLER KUTTNER 


Thus a bacteriophage, active against the susceptible variant 
of no. 18, had been extracted after prolonged incubation from 
the resistant variant. Under the heading, variations in typhoid 
strains, above, it will be noted that this strain had become resis¬ 
tant to lytic action without exposure to the lytic agent. The 
streak from the bottle of 18D showed that it had become con¬ 
taminated with a Staphylococcus alhus and a diphtheroid and it 
was thought that possibly the question of symbiosis might play 
a part in the production of bacteriophage from the organisms 
themselves. Carrere and Lisbon (1922) have recently had a 
similar idea, but this has now definitely been disproved, since 
pure cultures yield bacteriophage. 

At the time we obtained the above result, we had not read 
Bail's (1921) article of September 15, 1921, in which he stated 
that he had isolated a bacteriophage from 3 old broth cultures 
active against Flexner dysentery bacilli. He does not state 
what organism was originally inoculated into the old broth. In 
a more recent article, Otto and Munter (1921) have succeeded 
in isolating bacteriophage in 9 instances, active against several 
organisms of the typhoid-dysentery group, obtained from old 
broth cultures of Flexner, Mt. Desert and Shiga dysentery and 
typhoid bacilli, respectively. The broths varied from three 
weeks old to six months. Unfortunately, the authors do not 
state whether they reisolated the strain from the broth to see 
whether it was resistant to the bacteriophage produced in the 
fluid in which it was growing, nor in case the bacteriophage was 
obtained in an old typhoid culture, for instance, whether the 
lytic fluid was active against the homologous typhoid strain, 
or only against other typhoid strains. It would also be of 
interest to know whether the broth was inoculated with old 
stock cultures, or recently isolated ones. 

We have succeeded in obtaining another bacteriophage from 
a two months old typhoid culture (no. 18 susceptible) active 
against Shiga dysentery, but apparently against no other or¬ 
ganism. We have not tried to date, to see whether by allowing 
it to act on Shiga dysentery for a few generations, we could obtain 
a bacteriophage active against typhoid. Culture no. 18 has been 
isolated for a year and a half. 


BACTERIOPHAGE PHENOMENA 


83 


The most interesting results in regard to obtaining bacterio¬ 
phage directly from the bacteria themselves have been obtained 
by Callow in this laboratory, and are about to be published. 
She has found that the filtrates of certain strains of staphylococci 
prepared as described below, added to young broth cultures of 
certain other strains of staphylococci, proved lytic. 

The filtrates were obtained by growing staphylococci on agar plates 
for eighteen hours, washing off the growth in sterile broth, or better, in 
distilled water containing 0.02 per cent sodium hydroxide, shaking the 
emulsion for thirty minutes to an hour, and then filtering through a 
Berkefeld filter. The filtrate thus obtained was active for 1 or more 
strains of staphylococci, but in no instance against the homologous 
strain. It was thus possible to wash the lytic agent directly off certain 
strains of staphylococci from comparatively young cultures active 
against other strains. Miss Callow has obtained the same results with 
filtrates of young broth cultures, but the results have been much less 
constant than with the washings from the agar cultures. In the broth 
cultures also the filtrate was only in very rare instances, active against 
the homologous strain originally inoculated into the broth, and this 
result was only obtained with older broth cultures. The strains used by 
Miss Callow in these experiments had been isolated from boils for a 
period of three or four months and had been transplanted frequently. 
Miss Callow had not tried the method of washing off agar growths 
with older stock cultures of staphylococci, so that we cannot state that 
old as well as recently isolated strains produce a bacteriophage principle 
by this method. 

A small number of experiments have been done using the method 
originated by Miss Callow with strains of typhoid and dysentery 
bacilli, but to date without success. A variety of strains were 
tried. No. 18 susceptible and 18D, the resistant variant, were 
used, also the Shiga bacillus, which is generally agreed to be the 
most susceptible organism of the typhoid-colon-dysentery group. 
Another recently isolated strain of typhoid and a susceptible 
Bad . coli culture were also tried. Six hour, twenty-four hour and 
four day agar growths were shaken in alkaline solution, but none 
of the filtrates tested against a variety of cultures showed any 
lytic action. However, this work has been done very recently 
and most of the strians only tried once so that the results are 
not wholly conclusive. 


84 


ANN GAYLER KUTTNER 


Summary of isolation of bacteriophage from bacteria themselves 

It can be stated that in the hands of four different workers, 
Bail, Otto and Munter, Callow and ourselves, it has been possible 
to isolate a bacteriophage from the bacteria themselves. With 
the typhoid-dysentery-colon group, this can most easily be 
done with old broth cultures. With staphylococci, the best 
results are obtained by washing off young agar cultures. The 
bacteriophages thus obtained are in the results obtained by 
Callow and ourselves, not usually active against the strain with 
which the broth or agar was originally inoculated. In the case 
of the bacteriophage, active against Shiga bacilli, obtained 
from an old typhoid culture, the typhoid culture had been iso¬ 
lated for a period of over a year, and been constantly used as a 
control and never given any signs of spontaneous lysis. 

A recent paper by Lisbon and Carrere (1922) is of interest in 
this connection. 

These authors state that they have been able to obtain a bacterio¬ 
phage active against Shiga dysentery by inoculating a Shiga broth 
culture with a recently isolated Bad. coli. After incubating this mixed 
culture for a variable length of time, the broth is filtered. This filtrate 
is then added to a culture of Shiga bacilli and carried along for three or 
four generations. A bacteriophage active against the Shiga bacilli 
is finally obtained. They obtained a similar result by using a strain of 
Proteus, but they do not state whether it was recently isolated or not. 

D’Herelle (1922) has answered Lisbon and Carriere by saying 
that since the strains of Bact. coli that they used were all recently 
isolated from stools and urine, the probability is that they were 
dealing with organisms that were carrying a bacteriophage, 
although apparently no evidence of lysis in the Bact. coli culture 
was observed. D’Herelle has described in his book (page 58) 
what he calls mixed cultures of bacteria and the ultramicroscopic 
virus in which the resistance of the bacteria is sufficient to pre¬ 
vent the formation of lytic colonies, and the virus is carried by 
what appear to be perfectly normal colonies. 

Bordet and Ciuca were the first workers to show that the lytic 
agent could be carried with a certain type of Bact. coli colony ob- 


BACTERIOPHAGE PHENOMENA 


85 


tained after the culture was exposed to lysis. We have found that 
in the case of typhoid and dysentery cultures, bacteriophage action 
always divided the culture into lytic-bearing and what appeared 
to be non-lytic normal colonies. We streaked one of these 
normal colonies for fifteen successive generations, as reported 
above, on agar without ever obtaining anything but normal 
colonies. We also fished some of these normal colonies to broth 
and tried out the supernatant fluid comparison with the super¬ 
natant fluid of broth fishings of lytic colonies, without obtaining 
any evidence that the lytic agent was carried by these normal 
colonies. We, therefore, concluded that only the colonies in 
which we could definitely see evidence of lysis—i.e., lytic 
colonies—carried the virus. 

In obtaining bacteriophage principles from old broth cultures 
and from agar washings of young growths of Staphylococcus 
(Callow) it seemed at first that we had definitely proved that 
the lytic material was derived from the bacteria themselves. 
But we realize now that the argument will not be conclusive 
until we are able to demonstrate lytic activity in old broth cul¬ 
tures of old laboratory strains, or perhaps by the aid of the Bar¬ 
ber single cell isolation method. In both instances where we 
obtained lytic activity with old broth cultures, the culture used 
had in one instance been isolated six months and in the other 
over one year, but it was originally derived from a typhoid case 
in which the feces were shown to contain a potent bacterio¬ 
phage. We have also described in detail how this culture fluc¬ 
tuated in resistance to the Olsen bacteriophage. The defenders 
of the filtrable virus theory would see proof in this for their claim 
that an apparently normal culture can carry a bacteriophage 
without giving any evidence of it, since this culture six months 
after isolation suddenly became resistant and when inoculated 
into broth, after prolonged incubation, produced a bacterio¬ 
phage. The sudden resistance of this strain might, however, 
also be explained more simply by saying that in the course of the 
transplantation a series of resistant bacilli, which we know to 
exist in every culture, happened to be transferred for successive 
transplants until nothing but resistant bacilli were present. 


86 


ANN GAYLER KUTTNER 


The German writers do not give the history of the strains used 
by them. Miss Callow to date has only been successful with 
strains of recent isolation. We cannot, therefore, at the present 
time, completely exclude the possibility that apparently normal 
bacteria may carry an ultramicroscopic parasite. 

IV. RESISTANCE OF THE LYTIC AGENT 

Acetone resistance (Olsen bacteriophage ) 

A great many workers have tried to disprove the living virus 
theory in regard to bacteriophage action, notably Kabeshima, 
by pointing out the great resistance of the lytic agent. In our 
first attempt we were unable to confirm the method devised by 
Kabeshima for isolating the lytic material by precipitation with 
acetone. Recently, however, we have been able to demonstrate 
lytic activity in an acetone precipitate. Instead of starting 
with the simple dissolved culture filtrate obtained by the action 
of the Olsen bacteriophage on a typhoid culture, we concentrated 
the filtrate to one-tenth its volume by evaporation with a fan. 

To 50 cc. of this concentrated lytic broth 150 cc. of acetone were 
added and the mixture was allowed to stand at room temperature and 
shaken from time to time. At the end of 48 hours the acetone was 
evaporated off until only a syrupy brown liquid remained. This was 
centrifuged, and a small amount of yellow precipitate obtained. The 
supernatant fluid was pipetted off and the precipitate partially re¬ 
dissolved in salt solution. Both the salt solution solute thus obtained 
and the supernatant fluid proved active. 

It may be argued that the only reason that this concentrated 
lytic broth was able to withstand the forty-eight-hour exposure 
to acetone was because it was protected by the concentration 
of the proteins in the broth; but in any case, it shows a fairly 
high degree of resistance for the lytic agent. We concentrated 
the lytic precipitate in the first place to see if we could obtain a 
lytic agent of much greater potency. In one experiment the 
lytic titer was very much increased after concentration, but in 
subsequent tests the increase in titer was not so striking. The 


BACTERIOPHAGE PHENOMENA 


87 


Olsen bacteriophage is active in a dilution of 1:1,100,000, with 
most batches, without being concentrated. 

We also precipitated 1 volume of the concentrated lytic broth with 
9 volumes of acetone, and centrifuged the emulsion thus obtained, and 
redissolved the precipitate in salt solution. The precipitate dissolved 
very readily. The solution thus obtained, however, showed no lytic 
activity. 

Typhoid dissolved culture filtrate (Olsen bacteriophage), if 
concentrated at one-tenth its volume, will resist exposure to 3 
volumes of acetone for forty-eight hours. If, however, exposed 
to 9 volumes of acetone for a very short period, it is destroyed. 

Resistance to alcohol precipitation 

D’Herelle, in his book, states that by means of alcohol precipi¬ 
tation he has been able to separate the filtrable virus from the 
enzyme by which it acts. 

If the lytic filtrate is exposed to 95 per cent alcohol for forty-eight 
hours the virus is destroyed, but the enzyme by which the virus does 
its work is still active. If, therefore, the redissolved precipitate ob¬ 
tained by adding 9 volumes of alcohol to one of lytic broth, is added to a 
turbid broth culture of a susceptible organism the clarification of the 
broth takes place, but the lytic agent is no longer transmissible in series, 
and no lytic colonies are obtained on streaking the dissolved cultures. 
The virus according to d’Herelle resists exposure shorter than forty-eight 
hours. 

We have not to date been able to verify this particular experiment, 
but we have confirmed the extreme resistance of the lytic agent to alcohol 
precipitation. Exposure of 1 volume of concentrated lytic broth to 
12 volumes of absolute alcohol for two hours did not destroy its activity. 
Miss Callow, using saline washings of agar cultures which contain a 
minimum of protective protein, has noted resistance to precipitation 
with alcohol for twenty-four hours. 

The lytic agent, therefore, is more resistant than any form of life with 
which we are familiar, with exception of certain spores and an ultrami¬ 
crobe parasitic on tobacco cited by d’Herelle. 


88 


ANN GAYLER KUTTNER 


We have also verified the observation of other workers that the lytic 
agent is resistant to exposure to 50 per cent glycerol and chloroform. 
It is interesting to note that Rettger in 1905 had found that autolysins 
of the bacteria were more resistant to 10 per cent chloroform than to 
10 per cent toluol. We exposed the Olsen bacteriophage to 10 per 
cent chloroform and 50 per cent glycerol for a period of thirteen days 
without loss of activity. 

V. IS THE LYTIC AGENT ANTIGENIC? 

We have immunized 4 rabbits with typhoid cultures dissolved 
by the action of the Olsen bacteriophage. All the rabbits were 
injected intravenously and stood the injections well. One rabbit 
received altogether ten injections, one six injections, the other two 
received four injections at intervals of about four days. We were 
never able to develop an antilytic serum of the potency described 
by Bordet and Ciuca. 

In the following experiment rabbit 1052 was bled after three days 
after the eighth injection, and rabbit 1098 was bled four days after the 
sixth injection. The experiment was carried out as follows: Equal 
mixtures of the immune serum and the Olsen bacteriophage were 
made, and added immediately and after thirty minutes’ incubation and 
after eighteen hours’ incubation. The test was set up as follows: 


TUBE 

IMMEDI¬ 

ATE 

BROTH 

CUL¬ 

TURE 

18 

SERUM-BACTERIOPHAGE MIXTURES 

READINGS 
EIGHTEEN HOURS 

PLATES AFTER 
EIGHTEEN HOURS 

1 

CC. 

2 

CC. 

0.5 

0.2 cc. mixture Olsen + 

+ + clumped 


2 

2 

0.5 

serum 1052, bled May 23 
0.2 cc. mixture Olsen + 

++ clumped 


3 

2 

0.5 

serum 1098, bled May 20 
0.2 cc. mixture Olsen + 

+ + 

•Lytic colonies 

4 

2 

0.5 

serum 1097 (normal) 

0.1 cc. mixture Olsen 

+ 


5 

2 

0.5 

0.2 cc. saline 

++ 

Normal growth 


(Turbidity equal to control, indicated by + + .) 


The same result was obtained after the mixtures of serum and Olsen 
bacteriophage were incubated for thirty minutes and for eighteen 
hours. 













BACTERIOPHAGE PHENOMENA 


89 


One other protocol is of interest in which the immune serum was 
tested against dilutions of the Olsen bacteriophage, and in which immune 
typhoid serum was used as a control in order to show that agglutina¬ 
tion alone did not interfere with lytic action. In this case the immune 
serum was added to the typhoid broth first and then the dilutions of 
the Olsen bacteriophage added. Serum was used from Rabbit 1052, 
which at that date had received nine injections. 


December 7, 1921 


TUBE 

BROTH 

CUL¬ 

TURE 

18 

SERUM 

OLSEN BACTERIOPHAGE 

SIX HOUR PLATES 

1 

CC. 

2 

CC. 

0.1 

0.2 cc. 1052 bled July 1 

0.1 cc. undiluted 

Many lytic 

2 

2 

0.1 

0.2 cc. 1052 bled July 1 * 

0.1 cc. 1:100 

colonies 
Many lytic 

3 

2 

0.1 

0.2 cc. 1052 bled July 1 

0.1 cc. 1:1000 

colonies 

Few transpar¬ 

4 

2 

0.1 

0.2 cc. immune typhoid 

0.1 cc. undiluted 

ent areas 
Many lytic 

5 

2 

0.1 

horse serum 

0.2 cc. immune typhoid 

0.1 cc. 1:100 

colonies 
Many lytic 

6 

2 

0.1 

horse serum 

0.2 cc. immune typhoid 

0.1 cc. 1:1000 

colonies 
Many lytic 

7 

2 

0.1 

horse serum 

0.1 cc. 1:1000 

colonies 

Many lytic 

8 

2 

0.1 


0.1 cc. 1:1000 

colonies 
Many lytic 

9 

2 

0.1 


0.3 cc. salt 

colonies 

Normal 

10 

2 

0.1 

0.2 cc. immune typhoid 


growth 

Normal 




horse serum 


growth 


The bacteriophage immune serum did not prevent lysis even if 
the bacteriophage was diluted 1:1000, although it was diminished 
in tube 3. Typhoid immune serum did not inhibit lysis. The other 
rabbit gave similar results. In some cases the antilytic serum 
tended to prevent the clearing up of the bacterial emulsion, but on 
plating lytic colonies were obtained in every instance. We have 
not, therefore, been able, like Bordet and Ciuca, to produce an 
antilytic serum of sufficient potency to prevent lytic action 














90 


ANN GAYLER KUTTNER 


permanently. We must agree with cTHerelle and Bail that the 
inhibition of lysis with immune sera prepared by the inoculation 
of large amounts of dissolved culture filtrate into rabbits is not 
complete. We have not found that normal rabbit sera inter¬ 
fere with lysis. 

Protection experiments 

It has been reported that guinea-pigs can be protected against 
an M.L.D. of culture by the injection of the homologous dis¬ 
solved culture filtrate, and many workers have used typhoid and 
staphylococci bacteriophages in treating human cases. 

We have performed experiments in which we have injected bacterio¬ 
phage before and after the infection had begun, and have not found that 
the injection of lytic filtrate had any very striking advantages over the 
injections of sterile broth. The M.L.D. of the typhoid culture used 
was large, the washings of one eighteen-hour agar slant of typhoid 
being necessary to kill a pig of between 200 and 300 grams. Smaller 
doses were tried, but failed to kill the control pigs regularly. In the 
final experiment two pigs were used for each step, a light pig and a heavy 
pig as far as possible in pairs, the heavier pair in each case being used 
for controls. Eight pigs were used altogether; 4 pigs were injected with 
the M. L. D. of culture at 10:30 a.m. After four hours 2 of these pigs 
were injected with 2 cc. of sterile broth each, the other 2 were injected 
ydth 2 cc. of the homologous typhoid dissolved culture filtrate (Olsen). 
In the other pigs the bacteriophage and the sterile broth were inoculated 
first, and after an interval of four hours the M. L. D. of culture was 
injected. The cultures for the animals which received the lytic agent 
and broth first were stored on ice during the interim, to prevent 
further growth. All the injections were made intraperitoneally. 


January 19, 1922. Animals in which the culture was injected first 



b* 

o 

WEIGHT 

M. L. D. 
TYPHOID 
CULTURE 

TIME 

BROTH 

OR 

BACTERIOPHAGE 

TIME 

RESULT 

Controls 

364 

gms. 

330 

1 slant no. 18 

a.m. 

10:30 

2 cc. broth 

p.m. 

2:30 

Dead 1/20/22, 9 a.m. 


363 

245 

1 slant no. 18 

10:30 

2 cc. broth 

2:30 

Dead 1/20/22, 9 a.m. 

Bacterio¬ 

369 

250 

1 slant no. 18 

10:32 

2 cc. Olsen 

2:32 

Dead 1/20/22,9 a.m. 

phage 

362 

180 

1 slant no. 18 

10:32 

2 cc. Olsen 

2:32 

Dead 1/19/22,10 p.m. 















BACTERIOPHAGE PHENOMENA 


91 


Animals in which the bacteriophage was injected first 



Ck 

o' 

WEIGHT 

BROTH 

OR 

BACTERIOPHAGE 

TIME 

M. L. D, 
TYPHOID 
CULTURE 

TIME 

RESULT 

Controls 

400 

gms. 

270 

2 cc. broth 

a.m. 

10:35 

1 slant no. 18 

p.m. 

3:00 

Survived 


323 

210 

2 cc.broth 

10:35 

1 slant no. 18 

3:00 

Found dead 1/20/22 

Bacterio¬ 

325 

270 

2 cc. Olsen 

10:37 

1 slant no. 18 

3:05 

9 a.m. 

Survived 

phage 

394 

195 

2 cc. Olsen 

10:37 

1 slant no. 18 

3:05 

Died in 48 hours 


At 4:30 p.m., January 19, 1922, all the pigs were punctured and plates 
made of the exudate with the following result: 

No. 364. Good growth; normal typhoid colonies. 

No. 363. Good growth; normal typhoid colonies. 

No. 369. Many lytic colonies. 

No. 362. No growth. 

No. 400. Growth obtained; normal. 

No. 323. Growth obtained; normal. 

No. 325. Many lytic colonies. 

No. 394. No growth. 

Lysis was, therefore, going on actively in the peritoneum of the pigs 
325 and 369, which had received inoculations of bacteriophage. No 
growth was obtained from the other two bacteriophage pigs, 362 and 
394. Every pig that died was autopsied to make sure that it had 
not died from any other cause, and typhoid bacilli were isolated 
from the peritoneal cavity of each. Only two pigs out of the eight 
survived: one that had been injected with 2 cc. bacteriophage and one 
that had been injected with 2 cc. of sterile broth four hours before the 
injection of the M.L.D. of typhoid bacilli. 

Conclusions from animal experimentation 

We have not been able to confirm the opinion of other workers 
that the protective action of dissolved culture filtrate is very 
striking. We were unable to show any very definite advantage 
in the case of the Olsen bacteriophage over the injection of 















92 


ANN GAYLER IOJTTNER 


sterile extract broth. Bordet and Ciuca obtained definite 
protection in guinea-pigs against injections of Bact.coli. Since 
guinea-pigs are not susceptible to infections either with typhoid 
or Bad. coli and death in both cases is probably of toxic origin, 
conclusions as to the value of bacteriophage treatment for 
human beings cannot be drawn from these experiments. 

Study of the leucocytic exudates obtained from guinea pigs in 
protection experiment 

The peritoneal exudates from all the pigs that died were collected, 
twice the volume of sterile broth added, and the tubes left standing at 
room temperature for forty-eight hours. At the end of this time they 
were plated with the following results: 

No. 364. Small number of lytic colonies. 

No. 363. Many lytic colonies. 

No. 369. No lytic colonies, pure typhoid. 

No. 362. No lytic colonies, pure typhoid. 

No. 323. No lytic colonies, pure typhoid. 

No. 394. No lytic colonies, pure typhoid. 

Thus the exudates from the two control pigs showed lytic colonies, 
whereas the exudates from the pigs that had been injected with bacterio¬ 
phage which had previously shown lytic colonies (no. 369), after stand¬ 
ing at room temperature showed nothing but resistant types. 

The tubes were kept on ice from January 22 to January 24 and 
then heated at 59° for thirty minutes, and tested for sterility. These 
exudates were set up against the stock strain no. 18 as follows: 


TUBE 

AMOUNT 

OP 

BROTH 

CUL¬ 

TURE 

18 

PERITONEAL EXUDATES 

PLATES AFTER SEVEN HOURS 

1 

CC. 

2 

CC. 

0.1 

0.5 cc. no. 364 

Few transparent areas 

2 

2 

0.1 

0.5 cc. no. 363 

Transparent areas 

3 

2 

0.1 

0.5 cc. no. 369 

Little growth, 1 lytic colony 

4 

2 

0.1 

0.5 cc. no. 362 

Little growth, 2 normal colonies 

5 

2 

0.1 

0.5 cc. no. 323 

Normal growth 

6 

2 

0.1 

0.5 cc. no. 394 

No growth obtained 

7 

2 

0.1 

Salt solution 

Normal growth 










BACTERIOPHAGE PHENOMENA 


93 


As already stated under the section on the production of 
bacteriophage by the method of Bordet and Ciuca, it was possible 
to produce a lytic exudate by a single injection of typhoid bacilli 
intraperitoneally into guinea-pigs. 

VI. IS THE LYTIC PHENOMENON A FACTOR IN RECOVERY FROM 

INFECTION? 

Occurrence of bacteriophage in carrier stools 

It seemed of interest in view of the claims made for the bene¬ 
ficial results obtained by d’Herelle in the treatment of dysentery 
cases with bacteriophage, and by others in the treatment of 
boils with staphylococcus lytic agent, to determine the incidence 
of bacteriophage in carrier stools. 

Through the courtesy of Dr. Krumwiede of the Research Labora¬ 
tory of the Health Department, we were able to obtain carrier 
stools at frequent intervals, and also in one instance a specimen 
of blood from one of the carriers. 

We obtained carrier stools from two typhoid carriers of long standing, 
Mary Mallon and Mary Newton, residing at the Riverside Hospital, 
at weekly intervals for a period of six weeks. The fecal specimens were 
plated on Endo’s medium and the homologous strain of typhoid isolated. 
The proportion of typhoid colonies in the stool of Mallon was con¬ 
sistently less than with Newton. The plates were always carefully 
examined with the microscope but in no instance were any lytic or ab¬ 
normal colonies observed. 

The fecal suspensions were treated in two ways, the first time the 
specimen was received: in one case the suspension was thoroughly shaken 
and filtered immediately, in the other it was shaken and then incubated 
for four hours and filtered. The filtrates thus obtained from Newton 
and Mallon were tested against the homologous strain of typhoid and 
also against the stock strain of typhoid no. 18, since it was thought that 
the typhoid strain in these carrier stools might be resistant. 


94 


ANN GAYLER KUTTNER 


February 7, 1922 


TUBE 

AMOUNT 

OP 

TYPHOID 

CULTURE 

STOOL FILTRATE 

TWO HOUR PLATES 


BROTH 




CC. 




1 

2 

Mallon 

1 cc. Mallon shaken filtrate, Feb- 

Many lytic colonies 




ruary 1, 1922 


2 

2 

No. 18 

1 cc. Mallon shaken filtrate, Feb- 

Normal growth 




ruary 1, 1922 


3 

2 

Mallon 

1 cc. Mallon shaken filtrate, incu- 

Many lytic colonies 




bated 4 hours, February 1 


4 

2 

No. 18 

1 cc. Mallon shaken filtrate, incu¬ 

Normal growth 




bated 4 hours, February 1 


5 

2 

Newton 

1 cc. Newton shaken filtrate, Feb¬ 





ruary 1 


6 

2 

No. 18 

1 cc. Newton shaken filtrate, Feb¬ 


7 

2 

Newton 

ruary 1 

1 cc. Newton shaken filtrate, incu¬ 

> Normal growth 




bated 4 hours, February 1 


8 

2 

No. 18 

1 cc. Newton shaken filtrate, incu¬ 





bated 4 hours, February 1 


9 

2 

Mallon 

0.2 cc. Olsen bacteriophage 

Many lytic colonies 

10 

2 

Newton 

0.2 cc. Olsen bacteriophage 

Suspicious colonies 





but not definitely 
lytic 

11 

2 

No. 18 

0.2 cc. Olsen bacteriophage 

No growth 

12 

2 

Mallon 

Salt solution 

1 

13 

2 

Newton 


\ Normal growth 

14 

2 

No. 18 


1 


The stool filtrate of Mallon was definitely lytic for the homologous 
strain of typhoid, but not for the stock strain no. 18. The Mallon 
strain of typhoid was not resistant to the Olsen bacteriophage. The 
filtrate filtered immediately after shaking was as good as the one filtered 
after four hours’ incubation. The filtrate from Newton had no action 
on the homologous typhoid strain nor on the stock strain no. 18. 

The specificity of the Mallon typhoid strain was tried out as shown 
in the table on page 95. 

The tubes were incubated for one hour and then held at room tempera¬ 
ture overnight, and plated. The filtrate was again active against the 
homologous strain of typhoid and against Shiga, but against none of 
the other cultures tried. 














BACTERIOPHAGE PHENOMENA 


95 


Specificity of the Mallon filtrate, February 1,1922 


TUBE 

AMOUNT 

OP 

BROTH 

CULTURE 

FIL¬ 

TRATE 

PLATED AFTER EIGHTEEN HOUR8 AT 
ROOM TEMPERATURE 

1 

CC. 

2 

0.1 cc. Mallon typhoid 

0.5 

Few lytic colonies 

2 

2 

0.1 cc. no. 18 typhoid 

0.5 

Normal growth 

3 

2 

0.1 cc. Newton typhoid 

0.5 

Normal growth 

4 

2 

0.1 cc. Rawlings stock 

0.5 

Normal growth 

5 

2 

0.1 cc. Shiga 

0.5 

Reduced growth, 1 ‘appearances’’ 

6 

2 

0.1 cc. Mt. Desert 

0.5 

No lytic colonies 

7 

2 

0.1 cc. Typhi Murium 

0.5 

No lytic colonies 


Tube 1 in the last experiment was heated at 56° for thirty minutes 
to kill the resistant forms, and then the supernatant fluid tried against 
four typhoid strains to see if the potency of the Mallon bacteriophage 
was increased in the second generation. 


February 11, 1922 


TUBE 

AMOUNT 

OP 

BROTH 

TYPHOID 

CULTURE 

SUPERNATANT FLUID 

TWO HOUR PLATES 

1 

CC. 

2 

Mallon 

0.5 cc. tube 1, February 9 

Lytic colonies 

2 

2 

No. 18 

0.5 cc. tube 1, February 9 

Normal growth 

3 

2 

Newton 

0.5 cc. tube 1, February 9 

Transparent areas 

4 

2 

Rawlings stock 

0.5 cc. tube 1, February 9 

Normal growth 


In the second generation the Mallon bacteriophage had extended its 
activity and was lytic for the Newton strain of typhoid, as well as for 
the homologous strain. 

Bacteriophage in a carrier stool active against a homologous BacL 

coli culture 

Repeated tests showed that the filtrates from the stool of the 
carrier Newton had no action on the homologous or other typhoid 
strains. It seemed of interest, therefore, to determine whether 
or not the Newton stool might possibly contain a bacteriophage 
active against some other organism present in the fecal flora. 
We consequently isolated three different types of lactose-ferment¬ 
ing organisms occurring on Endo plates of the Newton stool, to 

















96 


ANN GAYLER KUTTNER 


Russell’s medium and tried the Newton filtrate against them 
with the following result: 


February 12, 1922 


TUBE 

I AMOUNT OF 

1 BROTH 

CULTURE 

STOOL FILTRATE 

AND 

CONTROLS 

TWO HOUR PLATES 

EIGHTEEN HOUR 
PLATES 


CC. 





1 

2 

Newton ty¬ 
phoid 

4 

1 cc. Newton fil¬ 
trate, February 
10 

Normal growth 

Normal growth 

2 

2 

Newton ty¬ 
phoid 

1 cc. salt 

Normal growth 

Normal growth 

3 

2 

Newton coli 
(1) 

1 cc. Newton fil¬ 
trate, February 
10 

Normal growth 

Normal growth 

4 

2 

Newton coli 
(1) 

1 cc. salt 

Normal growth 

Normal growth 

5 

2 

Newton coli 
(2) 

1 cc. Newton fil¬ 
trate, February 
10 

Reduced growth 
(“appear¬ 
ances”) 

No growth 

6 

2 

Newton coli 
(2) 

1 cc. salt 

Normal growth 

Normal growth 

7 

2 

Newton coli 
(3) 

1 cc. Newton fil¬ 
trate, February 
10 

Normal growth 

Normal growth 

8 

2 

Newton coli 
(3) 

1 cc. salt 

Normal growth 



By setting up a second generation from tube 5 definite lytic colonies 
were obtained with this particular strain. A bacteriophage active 
against Bad. coli (2) has been obtained from the stool of Newton 6 
successive times from specimens obtained at 1 week intervals. The 
growth of this susceptible Bad. coli is very characteristic; it forms a 
large, coarse colony, and in broth has a tendency to sediment out, 
leaving the supernatant fluid clear. The Newton filtrates have not 
proved active against the other types of Bad. coli present in the Newton 
stool. 

Each specimen of Newton received was plated and contained in 
every instance a large number of typhoid colonies. The colonies of 













BACTERIOPHAGE PHENOMENA 


97 


Bad. coli (2) were much less numerous, and in two out of the 6 specimens 
could not be found on the plates. 

We have not studied the occurrence of bacteriophage consistently in a 
sufficient number of carriers to state in what proportion of carriers bac¬ 
teriophages active against the homologous typhoid strain occur. 

Conclusions on work with carrier stools 

We have been able to demonstrate a bacteriophage active 
against the homologous strain of typhoid in the stool filtrates 
of a carrier of over ten year’s standing and also in one that has 
been positive for nine months. In another typhoid carrier of 
long standing, we have shown a bacteriophage active against a 
certain strain of Bad. coli occurring in the fecal flora. Speci¬ 
mens from both these carriers consistently contained these 
particular bacteriophages over a period of six weeks. The pro¬ 
portion of the susceptible organisms in the stool containing 
bacteriophage seemed to be reduced, and in two instances the 
susceptible organism could not be found. The presence of bac¬ 
teriophage in carrier stools may possibly explain the often 
observed fact, that carrier stools are sometimes negative. 

A specimen of blood obtained from one of the carriers (Newton) 
was examined for the presence of bacteriophage in the circulation, 
but none was found. 

It is worth reporting, furthermore, that the Ida Olsen from 
whom the Olsen bacteriophage used throughout this paper was 
isolated in November 1920 when she was in the convalescent 
stage after typhoid, has also developed into a chronic carrier. 
Her stools have been positive for typhoid for a period of one and 
one-half years. A specimen of feces has recently been obtained 
from her, but we have not so far been able to obtain a bacterio¬ 
phage from it. 

Discussion as to the nature of bacteriophage 

Evidence as to the nature of bacteriophage is still inconclusive. 
D’Herelle and his co-workers are convinced that all lytic 
phenomena are due to an ultramicroscopic virus, normally 


98 


ANN GAYLER KUTTNER 


parasitic on the bacteria of the intestinal tract, but capable by 
adaptation of attacking a large number of organisms. 

Kabeshima first brought forward the point of view that the 
resistance of the lytic agent was such as to rule out living proto¬ 
plasm, and that the whole manner of action of the bacteriophage 
suggested enzyme activity. It is obvous that the lytic agent 
cannot be classified as an enzyme as commonly defined, since 
it characteristically acts on living rather than dead cells. Further¬ 
more, there is no analogy in the literature of ferments for an 
enzyme which is quantitatively increased after acting on a 
substrate. 

Bordet and Ciuca’s observation that lytic activity could be 
demonstrated in the leucocytic exudate obtained in the peritoneal 
cavity of a guinea-pig by the injection of a normal bacterial 
culture, seemed to point away from the parasitic theory of bac¬ 
teriophage, indicating that the source of the lytic agent was 
probably in the bacteria themselves. In the same way, our 
own experiments in which we produced lysis of typhoid bacilli, 
transmittable in series by the action of extracts of normal tissue 
and of normal serum seems to diminish the likelihood of an ex¬ 
ternal parasite, and suggests that the bacteria themselves are 
the source of the lytic agent. But by far the most striking evi¬ 
dence that the bacterial cell itself secretes bacteriophage under 
certain circumstances, or liberates it when it disintegrates in 
a certain way, is the fact that the lytic agent can be demonstrated 
in old broth cultures in the case of typhoid and dysentery bacilli, 
and in alkaline washings of eighteen hour agar cultures, in the 
case of the staphylococcus. 

The high resistance to heat and various chemical reagents also 
renders d’Herelle’s theory less likely, but is in itself not suffi¬ 
cient evidence that the lytic agent is not a living thing, since 
as d’Herelle has pointed out, there are certain forms of life, such 
as the spores of B. subtilis , and an ultramicroscopic virus occur¬ 
ring on tobacco, that are equally resistant. 

Again, as an important argument against the filtrable virus 
conception, we may cite our own experiments as well as those of 
others in which the lytic principle was developed in old broth 


BACTEKIOPHAGE PHENOMENA 


99 


cultures of previously resistant strains, and has been obtained 
in salt solution washings of young agar cultures derived from 
normal colonies. These facts cannot be reconciled with the fil- 
trable virus theory without assuming that apparently resistant 
bacteria may remain carriers for generations of the infecting 
parasite. From this point of view, since all the cultures used 
in these experiments were originally derived from the animal 
body where they may have been exposed to bacteriophage action, 
it is possible that what we assume to be a normal colony, is only 
one which is infected to a less degree with the lytic agent. Under 
certain circumstances the bacteriophage may become disso¬ 
ciated from the bacteria, and then be demonstrable. Therefore, 
until the strains of bacteria used to show that bacteriophage 
develops spontaneously in the process of bacterial growth can 
be definitely shown to be free of a contaminating parasite by 
the use of the Barber single cell technique, it will not be possible 
to disprove conclusively the ultramicroscopic virus theory of 
bacteriophage. 

In the present state of our knowledge, however, taking all 
facts into consideration it seems to us more reasonable to assume 
the simpler hypothesis that the bacteriophage represents a 
secretion of the bacteria, produced under certain circumstances, 
and of the nature of an autolysin. This autolysin, usually 
liberated in old bacterial cultures, as a consequence of cell dis¬ 
integration acts as a catalyst which destroys the delicately ad¬ 
justed equilibrium occurring in actively growing cells between 
constructive forces and destructive forces, in favor of the latter. 
Solution of the bacterial cell consequently results and occurs 
in such a way that more of the autolysin is liberated. 

What the significance of the bacteriophage may be from a 
therapeutic point of view is difficult to estimate at the present 
time. The fact that a bacteriophage isolated from boils as 
shown by Callow, is usually not active against the homologous 
strain of staphylococcus, is not very encouraging. Also the 
isolation of bacteriophages active against the homologous strain 
of typhoid from chronic carriers and the development into a 
carrier of a convalescent case in which there was a typhoid 


100 


ANN GAYLER KUTTNER 


bacteriophage present, are not easy to correlate with the sup¬ 
posed therapeutic value of the bacteriophage. The data pre¬ 
sented are too limited to warrant any conclusions. Progress 
will probably be made by determining whether bacteriophage 
phenomena occur in other pathological conditions such as pneu¬ 
monia and meningitis. 

SUMMARY 

It has been shown that bacteriophage phenomena with mem¬ 
bers of the typhoid group can be initiated by means of a variety 
of agents: normal tissue extracts from guinea-pigs, and normal 
rabbit sera, and that the lytic agent is, therefore, not necessarily 
due to any pathological condition. Bacteriophage action has also 
been obtained with old typhoid broth cultures in the same way 
as reported by Bail with dysentery. The bacteriophages pro¬ 
duced in this way have usually not been active against the ho¬ 
mologous strain, but have been active against other strains of 
the typhoid-dysentery group. 

It is suggested that bacteriophage phenomena may possibly 
have played a part in the observations- made by previous workers 
on the bactericidal titer of normal rabbit serum for typhoid. 

High resistance reported by other observers of bacteriophage 
to acetone and to alcohol precipitation, and also to chloroform 
and glycerol have been confirmed. 

Attempts to prepare antilytic sera of sufficient potency to 
prevent lytic action completely, were unsuccessful. 

Intraperitoneal injections of bacteriophage into guinea pigs 
have not afforded any very definite protective action against one 
M.L.D. of typhoid bacilli. 

A bacteriophage principle active against the homologous strain 
of typhoid in two typhoid carriers has been demonstrated. In 
another typhoid carrier it has been shown that there was no 
bacteriophage present active against typhoid, but that the fil¬ 
trates consistently contained one active against a certain strain 
of Bad. coli. In one other case, where a bacteriophage active 
against the homologous strain of typhoid was isolated during 
the convalescent stage of the disease, the patient has developed 
into a chronic carrier. 


BACTERIOPHAGE PHENOMENA 


101 


REFERENCES 

Bail 1921 Wiener Klin. Woch., 34, 447-449. 

Bordet and Ciuca 1920 C. R. Soc. Biol., 83, 1293-1295. 

Bordet and Ciuca 1921a C. R. Soc. Biol., 84, 276-278. 

Bordet and Ciuca 1921b C. R. Soc. Biol., 84,278-279. 

Bordet and Ciuca 1921c C. R. Soc. Biol. 84, 748-750. 

Callow Unpublished data. 

Cantacuzene and Marie 1919 C. R. Soc. Biol., 82, 842-845. 

Carr^re and Lisbon 1922 C. R. Soc. Biol., February 18. 
d’Herelle 1917 C. R. Acad. d. sc., 165, 373. 

d’Herelle 1921 Le Bacteriophage—son role dans l’immunite, Masson et Cie, 
Paris. 

d’Herelle 1922 C. R. Soc. Biol., 86, 360-361. 

Eli ay a and Pozerski 1921 C. R. Soc. Biol., 84, 708-710. 

Gratia 1921a C. R. Soc. Biol., 84, 753-754. 

Gratia 1921b C. R. Soc. Biol., 85, 882. 

Kabeshima 1920 C. R. Soc. Biol., 83, 219-221. 

Kuttner 1921 Proc. Soc. Exp. Biol, and Med., 18, 158-163. 

Lisbon, Boulet and Carrere 1922 C. R. Soc. Biol., 86 , 340-342. 

Lisbon and Carrere 1922 C. R. Soc. Biol., 86, 569-570. [ 

Otto and Munter 1921 Deut. Med. Woch., 47, No. 52. |§f 

Petersen, W. F. 1922 Protein Therapy and Nonspecific Resistance, Mac¬ 
millan Co., New York, p. 102. 

Teague and McWilliams 1917 Jour. Immunol., 2, 167-184. 

Turro 1921 C. R. Soc. Biol., 84, 60-61. 

Twort 1907 Proc. Royal Soc. of London, Ser. 3, 79, 329. 

Twort 1915 Lancet, 189, Part II, 1241-1243. 

Wollstein 1921 Jour. Exper. Med., 34, 467-476. 







t 














VITA 


Ann Gayler Kuttner was born in New York City, September 
16, 1894. She was prepared for college at the Rogers Hall 
School, Lowell, Mass., and entered Bryn Mawr in 1911. She 
transferred from Bryn Mawr to Barnard and obtained the degree 
of B.S. in 1915 from Columbia University. From 1915 until 
1917 she held the position of laboratory assistant in the Research 
Laboratory of the New York Health Department. During Sep¬ 
tember of 1917 she went to France and became bacteriologist 
at the Red Cross Hospital No. 2, Paris. Later she transferred 
to the Laboratory of Base Hospital No. 1, Etretat, France, where 
she remained until the end of the war, returning to America 
with the Presbyterian Unit during February, 1919. During 
1919-1920 she studied at the College of Physicians and Surgeons, 
obtaining the degree of M.A. in the Department of Bacteriology. 
In 1920 she was appointed assistant in the Department of 
Bacteriology. 


















JOURNAL OF BACTERIOLOGY 

VOLUME VIII, NUMBER 1, JANUARY, 1923 



CONTENTS 



Robert Chambers. A Micromanipulator for the Isolation of Bacteria and the Dis¬ 


section of Cells . . . ....... 1 

George G. DeBord. Certain Phases of Nitrogenous Metabolism in Bacterial Cultures 7 

W. L. Holman. Device for Tubing Cooked Meat Medium...'. 47 

Ann Gayler Ktjttner. Bacteriophage Phenomena... 49 
























































